Heteroaryl compounds and uses thereof

ABSTRACT

The present invention provides compounds, pharmaceutically acceptable compositions thereof, and methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This present application is a divisional of U.S. patent application Ser.No. 15/635,954, filed Jun. 28, 2017 (now U.S. Pat. No. 10,189,794),which is a divisional of U.S. patent application Ser. No. 15/133,755,filed Apr. 20, 2016 (now U.S. Pat. No. 9,695,132), which is a divisionalof U.S. patent application Ser. No. 14/212,048, filed Mar. 14, 2014 (nowU.S. Pat. No. 9,321,786), which claims priority to U.S. ProvisionalPatent Application Ser. No. 61/793,113, filed Mar. 15, 2013, the entirecontents of each of which are incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to compounds useful as inhibitors ofprotein kinases. The invention also provides pharmaceutically acceptablecompositions comprising compounds of the present invention and methodsof using said compositions in the treatment of various disorders.

SEQUENCE LISTING

In accordance with 37 CFR 1.52(e)(5), the present specification makesreference to a Sequence Listing submitted electronically in the form ofa text file (entitled “Sequence_Listing.txt,” created on May 27, 2014,11 KB in size). The entire contents of the Sequence Listing are hereinincorporated by reference, with the intention that, upon publication(including issuance), this incorporated sequence listing will beinserted in the published document immediately before the claims.

BACKGROUND OF THE INVENTION

The search for new therapeutic agents has been greatly aided in recentyears by a better understanding of the structure of enzymes and otherbiomolecules associated with diseases. One important class of enzymesthat has been the subject of extensive study is protein kinases.

Protein kinases constitute a large family of structurally relatedenzymes that are responsible for the control of a variety of signaltransduction processes within the cell. Protein kinases are thought tohave evolved from a common ancestral gene due to the conservation oftheir structure and catalytic function. Almost all kinases contain asimilar 250-300 amino acid catalytic domain. The kinases may becategorized into families by the substrates they phosphorylate (e.g.,protein-tyrosine, protein-serine/threonine, lipids, etc.).

In general, protein kinases mediate intracellular signaling by effectinga phosphoryl transfer from a nucleoside triphosphate to a proteinacceptor that is involved in a signaling pathway. These phosphorylationevents act as molecular on/off switches that can modulate or regulatethe target protein biological function. These phosphorylation events areultimately triggered in response to a variety of extracellular and otherstimuli. Examples of such stimuli include environmental and chemicalstress signals (e.g., osmotic shock, heat shock, ultraviolet radiation,bacterial endotoxin, and H₂O₂), cytokines (e.g., interleukin-1 (IL-1)and tumor necrosis factor α (TNF-α)), and growth factors (e.g.,granulocyte macrophage-colony-stimulating factor (GM-CSF), andfibroblast growth factor (FGF)). An extracellular stimulus may affectone or more cellular responses related to cell growth, migration,differentiation, secretion of hormones, activation of transcriptionfactors, muscle contraction, glucose metabolism, control of proteinsynthesis, and regulation of the cell cycle.

Many diseases are associated with abnormal cellular responses triggeredby protein kinase-mediated events as described above. These diseasesinclude, but are not limited to, autoimmune diseases, inflammatorydiseases, bone diseases, metabolic diseases, neurological andneurodegenerative diseases, cancer, cardiovascular diseases, allergiesand asthma, Alzheimer's disease, and hormone-related diseases.Accordingly, there remains a need to find protein kinase inhibitorsuseful as therapeutic agents.

SUMMARY OF THE INVENTION

It has now been found that compounds of this invention, andpharmaceutically acceptable compositions thereof, are effective asinhibitors of one or more protein kinases. Such compounds have generalformula I:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R², R³, X¹, X², X³, X⁴, X⁵, G, Y, T, and q, is as defined anddescribed in embodiments herein. In certain embodiments, R¹ is a warheadgroup.

Compounds of the present invention, and pharmaceutically acceptablecompositions thereof, are useful for treating a variety of diseases,disorders or conditions, associated with abnormal cellular responsestriggered by protein kinase-mediated events. Such diseases, disorders,or conditions include those described herein.

Compounds provided by this invention are also useful for the study ofkinases in biological and pathological phenomena; the study ofintracellular signal transduction pathways mediated by such kinases; andthe comparative evaluation of new kinase inhibitors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Mass Modification of FGFR4 by I-1.

FIG. 2: Data showing that compound I-69 has prolonged duration of action(PDA) against pFGFR4 signalling in MDA-MB-453 cells consistent with theresynthesis rate of FGFR4.

FIG. 3: Data showing that compound I-1 has PDA against pFGFR4 signallingin MDA-MB-453 cells consistent with the resynthesis rate of FGFR4,whereas its noncovalent, reversible analog, I-234 does not have PDA.

FIG. 4: The amino acid sequence of FGFR4 (SEQ ID NO. 1).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS 1. General Description ofCompounds of the Invention

In certain embodiments, the present invention provides irreversibleinhibitors of FGFR4. In some embodiments, such compounds include thoseof the formulae described herein, or a pharmaceutically acceptable saltthereof, wherein each variable is as defined and described herein.

2. Compounds and Definitions

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic hydrocarbon that is completely saturated or that containsone or more units of unsaturation, but which is not aromatic (alsoreferred to herein as “carbocycle” “cycloaliphatic” or “cycloalkyl”),that has a single point of attachment to the rest of the molecule.Unless otherwise specified, aliphatic groups contain I-6 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain I-5aliphatic carbon atoms. In other embodiments, aliphatic groups containI-4 aliphatic carbon atoms. In still other embodiments, aliphatic groupscontain I-3 aliphatic carbon atoms, and in yet other embodiments,aliphatic groups contain I-2 aliphatic carbon atoms. In someembodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refersto a monocyclic C₃-C₆ hydrocarbon that is completely saturated or thatcontains one or more units of unsaturation, but which is not aromatic,that has a single point of attachment to the rest of the molecule.Exemplary aliphatic groups are linear or branched, substituted orunsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as(cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

As used herein, the term “bridged bicyclic” refers to any bicyclic ringsystem, i.e. carbocyclic or heterocyclic, saturated or partiallyunsaturated, having at least one bridge. As defined by IUPAC, a “bridge”is an unbranched chain of atoms or an atom or a valence bond connectingtwo bridgeheads, where a “bridgehead” is any skeletal atom of the ringsystem which is bonded to three or more skeletal atoms (excludinghydrogen). In some embodiments, a bridged bicyclic group has 7-12 ringmembers and 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Such bridged bicyclic groups are well known in theart and include those groups set forth below where each group isattached to the rest of the molecule at any substitutable carbon ornitrogen atom. Unless otherwise specified, a bridged bicyclic group isoptionally substituted with one or more substituents as set forth foraliphatic groups. Additionally or alternatively, any substitutablenitrogen of a bridged bicyclic group is optionally substituted.Exemplary bridged bicyclics include:

The term “lower alkyl” refers to a C₁₋₄ straight or branched alkylgroup. Exemplary lower alkyl groups are methyl, ethyl, propyl,isopropyl, butyl, isobutyl, and tert-butyl.

The term “lower haloalkyl” refers to a C₁₋₄ straight or branched alkylgroup that is substituted with one or more halogen atoms.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen, orphosphorus (including, any oxidized form of nitrogen, sulfur, orphosphorus; the quaternized form of any basic nitrogen or; asubstitutable nitrogen of a heterocyclic ring, for example N (as in3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as inN-substituted pyrrolidinyl)).

The term “unsaturated”, as used herein, means that a moiety has one ormore units of unsaturation.

As used herein, the term “bivalent C₁₋₈ (or C₁₋₆) saturated orunsaturated, straight or branched, hydrocarbon chain”, refers tobivalent alkylene, alkenylene, and alkynylene chains that are straightor branched as defined herein.

The term “alkylene” refers to a bivalent alkyl group. An “alkylenechain” is a polymethylene group, i.e., —(CH₂)_(n)—, wherein n is apositive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylenegroup in which one or more methylene hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

The term “alkenylene” refers to a bivalent alkenyl group. A substitutedalkenylene chain is a polymethylene group containing at least one doublebond in which one or more hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

As used herein, the term “cyclopropylenyl” refers to a bivalentcyclopropyl group of the following structure:

The term “halogen” means F, Cl, Br, or I.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic andbicyclic ring systems having a total of five to fourteen ring members,wherein at least one ring in the system is aromatic and wherein eachring in the system contains three to seven ring members. The term “aryl”is used interchangeably with the term “aryl ring”. In certainembodiments of the present invention, “aryl” refers to an aromatic ringsystem. Exemplary aryl groups are phenyl, biphenyl, naphthyl, anthracyland the like, which optionally includes one or more substituents. Alsoincluded within the scope of the term “aryl”, as it is used herein, is agroup in which an aromatic ring is fused to one or more non-aromaticrings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, ortetrahydronaphthyl, and the like.

The terms “heteroaryl” and “heteroar-”, used alone or as part of alarger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refer togroups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms;having 6, 10, or 14π electrons shared in a cyclic array; and having, inaddition to carbon atoms, from one to five heteroatoms. The term“heteroatom” refers to nitrogen, oxygen, or sulfur, and includes anyoxidized form of nitrogen or sulfur, and any quaternized form of a basicnitrogen. Heteroaryl groups include, without limitation, thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and“heteroar-”, as used herein, also include groups in which aheteroaromatic ring is fused to one or more aryl, cycloaliphatic, orheterocyclyl rings, where the radical or point of attachment is on theheteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl,benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Aheteroaryl group is optionally mono- or bicyclic. The term “heteroaryl”is used interchangeably with the terms “heteroaryl ring”, “heteroarylgroup”, or “heteroaromatic”, any of which terms include rings that areoptionally substituted. The term “heteroaralkyl” refers to an alkylgroup substituted by a heteroaryl, wherein the alkyl and heteroarylportions independently are optionally substituted.

As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclicradical”, and “heterocyclic ring” are used interchangeably and refer toa stable 5- to 7-membered monocyclic or 7-10-membered bicyclicheterocyclic moiety that is either saturated or partially unsaturated,and having, in addition to carbon atoms, one or more, preferably one tofour, heteroatoms, as defined above. When used in reference to a ringatom of a heterocycle, the term “nitrogen” includes a substitutednitrogen. As an example, in a saturated or partially unsaturated ringhaving 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, thenitrogen is N (as in 3,4-dihydro-2H pyrrolyl), NH (as in pyrrolidinyl),or ⁺NR (as in N-substituted pyrrolidinyl).

A heterocyclic ring can be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure and any ofthe ring atoms can be optionally substituted. Examples of such saturatedor partially unsaturated heterocyclic radicals include, withoutlimitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. Theterms “heterocycle”, “heterocyclyl”, “heterocyclyl ring”, “heterocyclicgroup”, “heterocyclic moiety”, and “heterocyclic radical”, are usedinterchangeably herein, and also include groups in which a heterocyclylring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings,such as indolinyl, 3H indolyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl, where the radical or point of attachment is on theheterocyclyl ring. A heterocyclyl group is optionally mono- or bicyclic.The term “heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aryl or heteroarylmoieties, as herein defined.

As described herein, certain compounds of the invention contain“optionally substituted” moieties. In general, the term “substituted”,whether preceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. “Substituted” applies to one or more hydrogens that areeither explicit or implicit from the structure (e.g.,

refers to at least

refers to at least

Unless otherwise indicated, an “optionally substituted” group has asuitable substituent at each substitutable position of the group, andwhen more than one position in any given structure is substituted withmore than one substituent selected from a specified group, thesubstituent is either the same or different at every position.Combinations of substituents envisioned by this invention are preferablythose that result in the formation of stable or chemically feasiblecompounds. The term “stable”, as used herein, refers to compounds thatare not substantially altered when subjected to conditions to allow fortheir production, detection, and, in certain embodiments, theirrecovery, purification, and use for one or more of the purposesdisclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(∘); —(CH₂)₀₋₄OR^(∘); —O(CH₂)₀₋₄R^(∘), —O—(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄CH(OR^(∘))₂; —(CH₂)₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which are optionallysubstituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which is optionallysubstituted with R^(∘); —CH═CHPh, which is optionally substituted withR^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which is optionally substituted withR^(∘); —NO₂; —CN; —N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘);—N(R^(∘))C(S)R^(∘); —(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘)₂; —(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄OC(O)R^(∘);—OC(O)(CH₂)₀₋₄SR^(∘), SC(S)SR^(∘); —(CH₂)₀₋₄ SC(O)R^(∘);—(CH₂)₀₋₄C(O)NR^(∘) ₂; —C(S)NR^(∘) ₂; —C(S)SR^(∘); —SC(S)SR^(∘),—(CH₂)₀₋₄OC(O)NR^(∘) ₂; —C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘);—C(O)CH₂C(O)R^(∘); —C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘); —(CH₂)₀₋₄S(O)₂R^(∘); —(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘); —S(O)₂NR^(∘) ₂;—(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂; —N(R^(∘))S(O)₂R^(∘);—N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘); —P(O)R^(∘) ₂; —OP(O)R^(∘)₂; —OP(O)(OR^(∘))₂; SiR^(∘) ₃; —(C₁₋₄ straight or branchedalkylene)O—N(R^(∘))₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(R^(∘))₂, wherein each R^(∘) is optionally substitutedas defined below and is independently hydrogen, C₁₋₆, aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(∘), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which is optionally substituted as definedbelow.

Suitable monovalent substituents on R^(∘) (or the ring formed by takingtwo independent occurrences of R^(∘) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(haloR^(●)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●),—(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●),—(CH₂)₀₋₂NR^(●) ₂, —NO₂, —SiR^(●) ₃, —OSiR^(●) ₃, —C(O)SR^(●), —(C₁₋₄straight or branched alkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(∘) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C iv, aliphatic which is substituted as defined below, oran unsubstituted 5-6-membered saturated, partially unsaturated, or arylring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which is optionally substitutedas defined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which is optionallysubstituted as defined below, unsubstituted —OPh, or an unsubstituted5-6-membered saturated, partially unsaturated, or aryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, NHR^(●), —NR^(●) ₂, or —NO₂, wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid ormalonic acid or by using other methods used in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate,lactobionate, lactate, laurate, lauryl sulfate, malate, maleate,malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,oleate, oxalate, palmitate, pamoate, pectinate, persulfate,3-phenylpropionate, phosphate, pivalate, propionate, stearate,succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate,undecanoate, valerate salts, and the like.

Salts derived from appropriate bases include alkali metal, alkalineearth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representative alkali oralkaline earth metal salts include sodium, lithium, potassium, calcium,magnesium, and the like. Further pharmaceutically acceptable saltsinclude, when appropriate, nontoxic ammonium, quaternary ammonium, andamine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and arylsulfonate.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures including the replacement of hydrogen by deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools, as probes in biological assays, or astherapeutic agents in accordance with the present invention. In someembodiments, the R¹ group comprises one or more deuterium atoms.

As used herein, the term “irreversible” or “irreversible inhibitor”refers to an inhibitor (i.e. a compound) that is able to be covalentlybonded to a target protein kinase in a substantially non-reversiblemanner. That is, whereas a reversible inhibitor is able to bind to (butis generally unable to form a covalent bond) the target protein kinase,and therefore can become dissociated from the target protein kinase, anirreversible inhibitor will remain substantially bound to the targetprotein kinase once covalent bond formation has occurred. Irreversibleinhibitors usually display time dependency, whereby the degree ofinhibition increases with the time with which the inhibitor is incontact with the enzyme. In certain embodiments, an irreversibleinhibitor will remain substantially bound to a kinase once covalent bondformation has occurred and will remain bound for a time period that islonger than the life of the protein.

Methods for identifying if a compound is acting as an irreversibleinhibitor are known to one of ordinary skill in the art. Such methodsinclude, but are not limited to, enzyme kinetic analysis of theinhibition profile of the compound with the protein kinase target, theuse of mass spectrometry of the protein drug target modified in thepresence of the inhibitor compound, discontinuous exposure, also knownas “washout,” experiments, and the use of labeling, such asradiolabelled inhibitor, to show covalent modification of the enzyme, aswell as other methods known to one of skill in the art.

One of ordinary skill in the art will recognize that certain reactivefunctional groups can act as “warheads.” As used herein, the term“warhead” or “warhead group” refers to a functional group present on acompound of the present invention wherein that functional group iscapable of covalently binding to an amino acid residue (such ascysteine, lysine, histidine, or other residues capable of beingcovalently modified) present in the binding pocket of the targetprotein, thereby irreversibly inhibiting the protein. It will beappreciated that the -L-Y group, as defined and described herein,provides such warhead groups for covalently, and irreversibly,inhibiting the protein. In certain instances, a “pro-warhead group” isused in place of a warhead groups. Such pro-warhead groups convert to awarhead group in vivo or in vitro.

As used herein, the term “inhibitor” is defined as a compound that bindsto and/or inhibits the target protein kinase with measurable affinity.In certain embodiments, an inhibitor has an IC₅₀ and/or binding constantof less about 50 μM, less than about 1 μM, less than about 500 nM, lessthan about 100 nM, or less than about 10 nM.

The terms “measurable affinity” and “measurably inhibit,” as usedherein, means a measurable change in FGFR4 activity between a samplecomprising a compound of the present invention, or composition thereof,and FGFR4, and an equivalent sample comprising FGFR4, in the absence ofsaid compound, or composition thereof.

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. Theterm “stable”, as used herein, refers to compounds which possessstability sufficient to allow manufacture and which maintains theintegrity of the compound for a sufficient period of time to be usefulfor the purposes detailed herein (e.g., therapeutic or prophylacticadministration to a subject).

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable herein includes that embodiment as any single embodimentor in combination with any other embodiments or portions thereof.

3. Description of Exemplary Compounds

According to one aspect, the present invention provides a compound offormula I,

-   or a pharmaceutically acceptable salt thereof, wherein:-   X¹ is —NR⁴, N, —CR⁴R^(4′), or —CR⁴;-   X² is —NR⁵, N, —CR⁵R^(5′), or —CR⁵;-   X³ is N or CR⁶;-   X⁴ is N or CR⁷;-   X⁵ is N, C, or CH; wherein at least one of X¹, X², X³, X⁴, or X⁵ is    N;-   G is H, O, OR, or N(R)(R);-   Ring A is an optionally substituted group selected from phenyl, a    3-8 membered saturated or partially unsaturated carbocyclic ring, a    5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms    independently selected from nitrogen, oxygen, or sulfur, a 4-7    membered saturated or partially unsaturated heterocyclic ring having    1-4 heteroatoms independently selected from nitrogen, oxygen, or    sulfur, or a 7-10 membered bicyclic saturated, partially unsaturated    or aryl ring;-   each R is independently hydrogen or an optionally substituted group    selected from C₁₋₆ aliphatic, phenyl, a 3-8 membered saturated or    partially unsaturated carbocyclic ring, a 4-7 membered heterocylic    ring having 1-4 heteroatoms independently selected from nitrogen,    oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring    having 1-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur; or-   two R groups on the same nitrogen are taken together with the    nitrogen atom to which they are attached to form a 4-7 membered    heterocylic ring having 0-2 additional heteroatoms independently    selected from nitrogen, oxygen, or sulfur, or a 4-7 membered    heteroaryl ring having 0-4 additional heteroatoms independently    selected from nitrogen, oxygen, or sulfur;-   R¹ is a warhead group; wherein R¹ is attached to an atom adjacent to    the atom where T is attached;-   each R² is independently —R, halogen, -haloalkyl, —OR, —SR, —CN,    —NO₂, —SO₂R, —SOR, —C(O)R, —CO₂R, —C(O)N(R)₂, —NRC(O)R,    —NRC(O)N(R)₂, —NRSO₂R, or —N(R)₂;-   R³ is hydrogen, C₂₋₆ alkenyl, —W-Cy, or C₁₋₆ alkyl, wherein the C₁₋₆    alkyl is optionally substituted with 1-3 groups independently    selected from halogen, —CN, oxo, —OR′, or —C(O)O(C₁₋₆ alkyl);-   W is absent or is a bivalent C₁₋₃ alkylene chain optionally    substituted with one or more R″ and wherein one methylene unit of W    is optionally replaced with —O—, —S—, or —NR′—;-   each R is independently hydrogen or C₁₋₆ alkyl;-   each R is independently halogen or C₁₋₆ alkyl, wherein the C₁₋₆    alkyl is optionally substituted with 1-3 groups independently    selected from halogen, —CN, oxo, or -OR^(x); Cy is phenyl, C₃₋₇    cycloalkyl, or a 3-7 membered monocyclic or 5-10 membered bicyclic    saturated, partially unsaturated, or heteroaryl ring having 1-3    heteroatoms independently selected from nitrogen, oxygen, or sulfur,    wherein Cy is optionally substituted with 1-3 R^(x);-   each R^(x) is independently H, —CN, oxo, —NH₂, C₁₋₆ alkyl, halogen,    —OR′, —N(R′)₂, —NHC(O)(C₁₋₆ alkyl), —C(O)N(R′)₂, —C(O)O(C₁₋₆ alkyl),    —NHSO₂(C₁₋₆ alkyl), or —SO₂N(R′)₂;-   or R³ is absent if not allowed by valence;-   each of R⁴ and R^(4′) is independently hydrogen or an optionally    substituted group selected from C₁₋₆ aliphatic, phenyl, a 3-8    membered saturated or partially unsaturated carbocyclic ring which    is optionally bridged, a 4-7 membered heterocylic ring having 1-4    heteroatoms independently selected from nitrogen, oxygen, or sulfur,    a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms    independently selected from nitrogen, oxygen, or sulfur, or a 7-10    membered bicyclic saturated, partially unsaturated or aryl ring,    which is optionally bridged;-   each of R⁵ and R^(5′) is independently —R, halogen, —OR, —SR, —CN,    —NO₂, —SO₂R, —SOR, —C(O)R, —CO₂R, —C(O)N(R)₂, —NRC(O)R,    —NRC(O)N(R)₂, —NRSO₂R, or —N(R)₂;-   Y is O or NR^(a);-   R^(a) is hydrogen or an optionally substituted C₁₋₆ aliphatic group;-   T is a covalent bond or a bivalent straight or branched, saturated    or unsaturated C₁₋₆ hydrocarbon chain wherein one or more methylene    units are optionally replaced by —O—, —S—, —N(R)—, —C(O)—, —OC(O)—,    —C(O)O—, —C(O)N(R)—, —N(R)C(O)—, —N(R)C(O)N(R)—, —S(O)—, —SO₂—,    —SO₂N(R)—, —N(R)SO₂—, or —N(R)SO₂N(R)—;-   q is 0-6; and-   each of R⁶ and R⁷ is independently —R, halogen, —OR, —SR, —CN, —NO₂,    —SO₂R, —SOR, —C(O)R, —CO₂R, —C(O)N(R)₂, —NRC(O)R, —NRC(O)N(R)₂,    —NRSO₂R, or —N(R)₂.

In certain embodiments, X¹ is —NR⁴. In certain embodiments, X¹ is N. Incertain embodiments, X¹ is —CR⁴R^(4′). In certain embodiments, X¹ is—CR⁴.

In certain embodiments, X² is —NR⁵. In certain embodiments, X² is N. Incertain embodiments, X² is —CR⁵R^(5′). In certain embodiments, X² is—CR⁵.

In certain embodiments, X³ is N. In certain embodiments, X³ is CR⁶.

In certain embodiments, X⁴ is N. In certain embodiments, X⁴ is CR⁷.

In certain embodiments, X⁵ is N. In certain embodiments, X⁵ is C. Incertain embodiments, X⁵ is CH.

In certain embodiments, G is H. In certain embodiments, G is O. Incertain embodiments, G is OR. In certain embodiments, G is N(R)(R).

In certain embodiments, G is OMe. In certain embodiments, G is NH₂.

In certain embodiments, Y is O. In certain embodiments, Y is NR^(a).

As defined generally above, Ring A is an optionally substituted groupselected from phenyl, a 3-8 membered saturated or partially unsaturatedcarbocyclic ring, a 5-6 membered monocyclic heteroaryl ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, a4-7 membered saturated or partially unsaturated heterocyclic ring having1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur,or a 7-10 membered bicyclic saturated, partially unsaturated or arylring.

In certain embodiments, Ring A is an optionally substituted phenylgroup. In some embodiments, Ring A is an optionally substituted a 3-8membered saturated or partially unsaturated carbocyclic ring. In someembodiments, Ring A is an optionally substituted 5-6 membered monocyclicheteroaryl ring having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, Ring A is anoptionally substituted 4-7 membered saturated or partially unsaturatedheterocyclic ring having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, Ring A is anoptionally substituted 7-10 membered bicyclic saturated, partiallyunsaturated or aryl ring.

In various embodiments, Ring A is cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, adamantyl, cyclooctyl, [3.3.0]bicyclooctanyl,[4.3.0]bicyclononanyl, [4.4.0]bicyclodecanyl, [2.2.2]bicyclooctanyl,fluorenyl, phenyl, naphthyl, indanyl, tetrahydronaphthyl, acridinyl,azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl,benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl,carbazolyl, NH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-A]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 377-indolyl, isoindolinyl, isoindolenyl,isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl,isoquinolinyl, isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl; −1,2,5oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, pteridinyl, purinyl, pyranyl, pyrazinyl,pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,1,2,5-thiadiazolyl, 1,3,4thiadiazolyl, thianthrenyl, thiazolyl, thienyl,thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl,triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl,1,3,4-triazolyl, or xanthenyl.

In certain embodiments, Ring A is phenyl, cyclohexyl, cyclohexenyl,cyclopentyl, cyclobutyl, cyclopropyl, pyridine, pyrmidine, pyrazine,pyridazine, pyrrole, pyrazole, piperidine, piperidin-one, pyrrolidine,tetrahydropyran, tetrahydrofuran, tetrahydrothiophene dioxide, orcyclobutene dione.

In certain embodiments, Ring A is an optionally substituted groupselected from phenyl, cyclohexyl, a 7-8 membered saturated or partiallyunsaturated carbocyclic ring, or a 4-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In various embodiments, Ring A is an optionally substituted groupselected from phenyl or cyclohexyl.

In certain embodiments, Ring A is substituted as defined herein. In someembodiments, Ring A is substituted with one, two, or three R² groupseach of which is independently selected. Exemplary substituents on RingA include Br, I, Cl, F, Me, —CF₃, —OMe, —OR, —N(R)₂, pyrazolyl,thiazolyl, piperidinyl, piperazinyl, or morpholinyl.

Exemplary Ring A groups are set forth below:

In another embodiment, each R² is independently —R.

In another embodiment, each R² is hydrogen.

In another embodiment, each R² is independently halogen, -haloalkyl,—OR, —SR, —CN, —NO₂, —SO₂R, —SOR, —C(O)R, —CO₂R, —C(O)N(R)₂, —NRC(O)R,—NRC(O)N(R)₂, —NRSO₂R, or —N(R)₂.

In certain embodiments, each R² is independently methyl, ethyl, propyl,i-propyl, F, Cl, Br, I, CF₃, piperidinyl, piperazinyl, morpholinyl,tetrahydropyridinyl, pyrazolyl, thiazolyl, or tetrazolyl.

In certain embodiments, each R² is independently —CH₃, —Cl, —F, —CF₃, or—OMe; or is selected from

In certain embodiments, Ring A is selected from

In various embodiments, R³ is hydrogen.

In various embodiments, R³ is C₂₋₆ alkenyl, —W-Cy, or C₁₋₆ alkyl,wherein the C₁₋₆ alkyl is optionally substituted with 1-3 groupsindependently selected from halogen, —CN, oxo, —OR′, or —C(O)O(C₁₋₆alkyl).

In certain embodiments, W is absent (i.e., W is a covalent bond). Incertain embodiments, W is a bivalent C₁₋₃ alkylene chain optionallysubstituted with one or more R″ and wherein one methylene unit of W isoptionally replaced with —O—, —S—, or —NR′—. In certain embodiments, Cyis phenyl wherein Cy is optionally substituted with 1-3 R^(x). Incertain embodiments, Cy is C₃₋₇ cycloalkyl wherein Cy is optionallysubstituted with 1-3 R^(x). In certain embodiments, Cy is a 3-7 memberedmonocyclic or 5-10 membered bicyclic saturated or partially unsaturatedheterocyclic having 1-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, wherein Cy is optionally substituted with1-3 R^(x). In certain embodiments, Cy is a 3-7 membered monocyclic or5-10 membered bicyclic heteroaryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, wherein Cy isoptionally substituted with 1-3 R^(x).

In certain embodiments, R³ is C₁₋₆ alkyl.

In certain embodiments, R³ is cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, oxetanyl, phenyl, piperidinyl, pyridinyl,pyrazolyl, thiazolyl, or pyridin-one-yl.

In certain embodiments, R³ is

In certain embodiments, R³ is absent.

In certain embodiments, each of R⁴ and R^(4′) is independently hydrogen.

In certain embodiments, each of R⁴ and R^(4′) is independently anoptionally substituted phenyl. In certain embodiments, each of R⁴ andR^(4′) is independently an optionally substituted C₁₋₆ aliphatic. Incertain embodiments, each of R⁴ and R^(4′) is independently a 3-8membered saturated or partially unsaturated carbocyclic ring. In otherembodiments, each of R⁴ and R^(4′) is independently an optionallysubstituted 4-7 membered heterocylic ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or a 5-6membered monocyclic heteroaryl ring having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In certain embodiments, each of R⁴ and R^(4′) is independently anoptionally substituted phenyl or a 3-8 membered saturated or partiallyunsaturated carbocyclic ring. In certain embodiments, each of R⁴ andR^(4′) is independently an optionally substituted phenyl.

In certain embodiments, each of R⁴ and R^(4′) is independently anoptionally substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, oxetanyl, phenyl, piperidinyl, pyridinyl, pyrazolyl,thiazolyl, or pyridin-one-yl.

In certain embodiments, each of R⁴ and R^(4′) is independently is ethyl,phenyl, cyclohexyl,

In certain embodiments, each of R⁵ and R^(5′) is independently —R.

In certain embodiments, each of R⁵ and R^(5′) is independently H. Incertain embodiments, both of R⁵ and R^(5′) are H. In certainembodiments, one of R⁵ and R^(5′) is H. In certain embodiments, each ofR⁵ and R^(5′) is independently H, or -Me.

In certain embodiments, each of R⁵ and R^(5′) is independently halogen,—OR, —SR, —CN, —NO₂, —SO₂R, —SOR, —C(O)R, —CO₂R, —C(O)N(R)₂, —NRC(O)R,—NRC(O)N(R)₂, —NRSO₂R, or —N(R)₂.

In some embodiments, R^(a) is hydrogen. In some embodiments, R^(a) isoptionally substituted C₁₋₆ aliphatic group.

In another embodiment, T is a covalent bond. In another embodiment, T isa bivalent straight or branched, saturated or unsaturated C₁₋₆hydrocarbon chain wherein one or more methylene units are optionallyreplaced by —O—, —S—, —N(R)—, —C(O)—, —OC(O)—, —C(O)O—, —C(O)N(R)—,—N(R)C(O)—, —N(R)C(O)N(R)—, —S(O)—, —SO₂—, —SO₂N(R)—, —N(R)SO₂—, or—N(R)SO₂N(R)—.

In another embodiment, T is a bivalent straight or branched, saturatedor unsaturated C₁₋₆ hydrocarbon chain wherein one or more methyleneunits are optionally replaced by —O—, —S—, —N(R)—, —C(O)—, —OC(O)—,—C(O)O—, —C(O)N(R)—, —N(R)C(O)—, —N(R)C(O)N(R)—, —N(R)SO₂—, or—N(R)SO₂N(R)—.

In another embodiment, T is a covalent bond or a bivalent straight orbranched, saturated or unsaturated C₁₋₆ hydrocarbon chain.

In certain embodiments, q is 0. In other embodiments, q is 1. In otherembodiments, q is 2-6.

In certain embodiments, the present invention provides a compound offormula I-a,

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R²,R³, R⁴, R⁵, R^(5′), R^(a), T, and q is as defined above and described inembodiments, classes and subclasses above and herein, singly or incombination.

In certain embodiments, the compound is of formula I-b:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R², R³, R⁴, T, and q is as defined above and described inembodiments, classes and subclasses above and herein, singly or incombination.

In certain embodiments, the invention provides a compound of formulaI-c:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R²,R³, R⁴, R^(a), and q is as defined above and described in embodiments,classes and subclasses above and herein, singly or in combination.

In certain embodiments, the invention provides a compound of formulaI-d:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R²,R³, R⁴, R^(a), and q is as defined above and described in embodiments,classes and subclasses above and herein, singly or in combination.

In other embodiments, the invention provides a compound of formula I-e:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R², R³, R⁵, R^(5′), R^(a), and q is as defined above and describedin embodiments, classes and subclasses above and herein, singly or incombination.

In other embodiments, the invention provides a compound of formula I-f:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R², R³, R⁵, R^(5′), R^(a), and q is as defined above and describedin embodiments, classes and subclasses above and herein, singly or incombination.

In certain embodiments, the invention provides a compound of formulaI-g:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R², R³, R⁴, R^(a), and q is as defined above and described inembodiments, classes and subclasses above and herein, singly or incombination.

In certain embodiments, the invention provides a compound of formulaI-h:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R², R³, R⁴, R^(a), and q is as defined above and described inembodiments, classes and subclasses above and herein, singly or incombination.

In certain embodiments, the invention provides a compound of formulaI-j:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R², R⁴, R^(a), and q is as defined above and described inembodiments, classes and subclasses above and herein, singly or incombination.

In certain embodiments, the invention provides a compound of formulaI-k:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R², R³, R⁴, R^(a), and q is as defined above and described inembodiments, classes and subclasses above and herein, singly or incombination.

In certain embodiments, the invention provides a compound of formulaI-n:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R², R³, R⁴, and q is as defined above and described in embodiments,classes and subclasses above and herein, singly or in combination.

In certain embodiments, the compound is of formula I-q:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,R¹, R², R³, R⁴, T, and q is as defined above and described inembodiments, classes and subclasses above and herein, singly or incombination.

In certain embodiments, the invention provides a compound selected fromTable 1 or Table 2:

TABLE 1

I-1

I-2

I-3

I-4

I-5

I-6

I-7

I-8

I-9

I-10

I-11

I-12

I-13

I-14

I-15

I-16

I-17

I-18

I-19

I-20

I-21

I-22

I-23

I-24

I-25

I-26

I-27

I-28

I-29

I-30

I-31

I-32

I-33

I-34

I-35

I-36

I-37

I-38

I-39

I-40

I-41

I-42

I-43

I-44

I-45

I-46

I-47

I-48

I-49

I-50

I-51

I-52

I-53

I-54

I-55

I-56

I-57

I-58

I-59

I-60

I-61

I-62

I-63

I-64

I-65

I-66

I-67

I-68

I-69

I-70

I-73

I-74

I-75

I-76

I-77

I-78

I-79

I-80

I-81

I-82

I-83

I-84

I-85

I-86

I-87

I-88

I-89

I-90

I-91

I-92

I-93

I-94

I-95

I-96

I-97

I-98

I-112

I-113

I-114

I-119

I-120

I-121

I-122

I-123

I-124

I-125

I-126

I-127

I-128

I-133

I-134

I-135

I-136

I-137

I-138

I-139

I-140

I-141

I-142

I-143

I-144

I-145

I-146

I-147

I-148

I-149

I-150

I-151

I-152

I-153

I-154

I-155

I-156

I-157

I-158

I-159

I-160

I-161

I-162

I-163

I-164

I-165

I-166

I-167

I-168

I-170

I-171

I-172

I-173

I-174

I-175

I-176

I-177

I-179

I-180

I-181

I-182

I-183

I-184

I-185

I-186

I-187

I-188

I-189

I-190

I-191

I-193

I-194

I-195

I-196

I-197

I-198

I-199

I-200

I-201

I-202

I-203

I-204

I-205

I-206

I-207

I-208

I-209

I-210

I-211

I-212

I-213

I-214

I-215

I-216

I-217

I-218

I-219

I-220

I-221

I-222

I-223

I-224

I-225

I-226

I-227

I-228

I-229

I-230

I-231

I-232

I-233

I-240

I-241

I-242

I-243

I-244

I-245

I-246

I-247

In certain embodiments, the invention provides a compound selected fromTable 2:

TABLE 2

I-71

I-72

I-99

I-100

I-101

I-102

I-103

I-104

I-105

I-106

I-107

I-108

I-109

I-110

I-111

I-117

I-115

I-116

I-129

I-130

I-131

I-132

I-169

In certain embodiments, the invention provides a compound selected from:

In some embodiments, the present invention provides a compound selectedfrom those depicted above, or a pharmaceutically acceptable saltthereof.

As defined generally above, the R¹ group of any of the formulae hereinis -L-Y, wherein:

-   -   L is a covalent bond or a bivalent C₁₋₉ saturated or        unsaturated, straight or branched, hydrocarbon chain, wherein        one, two, or three methylene units of L are optionally and        independently replaced by cyclopropylene, —NR—, —N(R)C(O)—,        —C(O)N(R)—, —N(R)SO₂—, —SO₂N(R)—, —O—, —C(O)—, —OC(O)—, —C(O)O—,        —S—, —SO—, —SO₂—, —C(═S)—, —C(═NR)—, —N═N—, or —C(═N₂)—;    -   Y is hydrogen, C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN, or a 3-10 membered monocyclic or bicyclic,        saturated, partially unsaturated, or aryl ring having 0-3        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, and wherein said ring is substituted with 1-4 R^(e)        groups; and    -   each R^(e) is independently selected from -Q-Z, oxo, NO₂,        halogen, CN, a suitable leaving group, or a C₁₋₆ aliphatic        optionally substituted with oxo, halogen, NO₂, or CN, wherein:        -   Q is a covalent bond or a bivalent C₁₋₆ saturated or            unsaturated, straight or branched, hydrocarbon chain,            wherein one or two methylene units of Q are optionally and            independently replaced by —N(R)—, —S—, —O—, —C(O)—, —OC(O)—,            —C(O)O—, —SO—, or —SO₂—, —N(R)C(O)—, —C(O)N(R)—, —N(R)SO₂—,            or —SO₂N(R)—; and        -   Z is hydrogen or C₁₋₆ aliphatic optionally substituted with            oxo, halogen, NO₂, or CN.

In certain embodiments, Lisa covalent bond.

In certain embodiments, L is a bivalent C₁₋₈ saturated or unsaturated,straight or branched, hydrocarbon chain. In certain embodiments, L is—CH₂—.

In certain embodiments, L is a covalent bond, —CH₂—, —NH—, —CH₂NH—,—NHCH₂—, —NHC(O)—, —NHC(O)CH₂OC(O)—, —CH₂NHC(O)—, —NHSO₂—, —NHSO₂CH₂—,—NHC(O)CH₂OC(O)—, or —SO₂NH—.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and one or twoadditional methylene units of L are optionally and independentlyreplaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—,—SO₂—, —OC(O)—, —C(O)O—, cyclopropylene, —O—, —N(R)—, or —C(O)—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —C(O)—, —NRC(O)—, —C(O)NR—,—N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—, and oneor two additional methylene units of L are optionally and independentlyreplaced by cyclopropylene, —O—, —N(R)—, or —C(O)—.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —C(O)—, and one or two additionalmethylene units of L are optionally and independently replaced bycyclopropylene, —O—, —N(R)—, or —C(O)—.

As described above, in certain embodiments, Lisa bivalent C₂₋₈ straightor branched, hydrocarbon chain wherein L has at least one double bond.One of ordinary skill in the art will recognize that such a double bondmay exist within the hydrocarbon chain backbone or is “exo” to thebackbone chain and thus forming an alkylidene group. By way of example,such an L group having an alkylidene branched chain includes—CH₂C(═CH₂)CH₂—. Thus, in some embodiments, Lisa bivalent C₂₋₈ straightor branched, hydrocarbon chain wherein L has at least one alkylidenyldouble bond. Exemplary L groups include —NHC(O)C(═CH₂)CH₂—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —C(O)—. In certain embodiments, Lis —C(O)CH═CH(CH₃)—, —C(O)CH═CHCH₂NH(CH₃)—, —C(O)CH═CH(CH₃)—,—C(O)CH═CH—, —CH₂C(O)CH═CH—, —CH₂C(O)CH═CH(CH₃)—, —CH₂CH₂C(O)CH═CH—,—CH₂CH₂C(O)CH═CHCH₂—, —CH₂CH₂C(O)CH═CHCH₂NH(CH₃)—, or—CH₂CH₂C(O)CH═CH(CH₃)—, or —CH(CH₃)OC(O)CH═CH—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —OC(O)—.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—,—SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—, and one or twoadditional methylene units of L are optionally and independentlyreplaced by cyclopropylene, —O—, —N(R)—, or —C(O)—. In some embodiments,L is —CH₂OC(O)CH═CHCH₂—, —CH₂—OC(O)CH═CH— or —CH(CH═CH₂)OC(O)CH═CH—.

In certain embodiments, L is —NRC(O)CH═CH—, —NRC(O)CH═CHCH₂N(CH₃)—,—NRC(O)CH═CHCH₂O—, —CH₂NRC(O)CH═CH—, —NRSO₂CH═CH—, —NRSO₂CH═CHCH₂—,—NRC(O)(C═N₂)—, —NRC(O)(C═N₂)C(O)—, —NRC(O)CH═CHCH₂N(CH₃)—,NRC(O)C(═CH₂)CH₂—, —CH₂NRC(O)—, —CH₂CH₂NRC(O)—, or—CH₂NRC(O)cyclopropylene-; wherein R is H or optionally substituted C₁₋₆aliphatic; and Y is hydrogen or C₁₋₆ aliphatic optionally substitutedwith oxo, halogen, NO₂, or CN.

In certain embodiments, L is —NHC(O)CH═CH—, —NHC(O)CH═CHCH₂N(CH₃)—,—NHC(O)CH═CHCH₂O—, —CH₂NHC(O)CH═CH—, —NHSO₂CH═CH—, —NHSO₂CH═CHCH₂—,—NHC(O)(C═N₂)—, —NHC(O)(C═N₂)C(O)—, —NHC(O)C(═CH₂)CH₂—, —CH₂NHC(O)—,—CH₂CH₂NHC(O)—, or —CH₂NHC(O)cyclopropylene-.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one triple bond. In certainembodiments, L is a bivalent C₂₋₈ straight or branched, hydrocarbonchain wherein L has at least one triple bond and one or two additionalmethylene units of L are optionally and independently replaced by—NRC(O)—, —C(O)NR—, —S—, —S(O)—, —SO₂—, —C(═S)—, —C(═NR)—, —O—, —N(R)—,or —C(O)—. In some embodiments, L has at least one triple bond and atleast one methylene unit of L is replaced by —N(R)—, —N(R)C(O)—, —C(O)—,—C(O)O—, or —OC(O)—, or —O—.

Exemplary L groups include —C≡C—, —C≡CCH₂N(isopropyl)-,—NHC(O)C≡CCH₂CH₂—, —CH₂—C≡C—CH₂—, —C≡CCH₂O—, —CH₂C(O)C≡C—, —C(O)C≡C—, or—CH₂OC(═O)C≡C—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein one methylene unit of L is replaced bycyclopropylene and one or two additional methylene units of L areindependently replaced by —C(O)—, —NRC(O)—, —C(O)NR—, —N(R)SO₂—, or—SO₂N(R)—. Exemplary L groups include —NHC(O)-cyclopropylene-SO₂— and—NHC(O)— cyclopropylene-.

As defined generally above, Y is hydrogen, C₁₋₆ aliphatic optionallysubstituted with oxo, halogen, NO₂, or CN, or a 3-10 membered monocyclicor bicyclic, saturated, partially unsaturated, or aryl ring having 0-3heteroatoms independently selected from nitrogen, oxygen, or sulfur, andwherein said ring is substituted with at 1-4 R^(e) groups, each R^(e) isindependently selected from -Q-Z, oxo, NO₂, halogen, CN, a suitableleaving group, or C₁₋₆ aliphatic, wherein Q is a covalent bond or abivalent C₁₋₆ saturated or unsaturated, straight or branched,hydrocarbon chain, wherein one or two methylene units of Q areoptionally and independently replaced by —N(R)—, —S—, —O—, —C(O)—,—OC(O)—, —C(O)O—, —SO—, or —SO₂—, —N(R)C(O)—, —C(O)N(R)—, —N(R)SO₂—, or—SO₂N(R)—; and, Z is hydrogen or C₁₋₆ aliphatic optionally substitutedwith oxo, halogen, NO₂, or CN.

In certain embodiments, Y is hydrogen.

In certain embodiments, Y is C₁₋₆ aliphatic optionally substituted withoxo, halogen, NO₂, or CN. In some embodiments, Y is C₂₋₆ alkenyloptionally substituted with oxo, halogen, NO₂, or CN. In otherembodiments, Y is C₂₋₆ alkynyl optionally substituted with oxo, halogen,NO₂, or CN. In some embodiments, Y is C₂₋₆ alkenyl. In otherembodiments, Y is C₂₋₄ alkynyl.

In other embodiments, Y is C₁₋₆ alkyl substituted with oxo, halogen,NO₂, or CN. Such Y groups include —CH₂F, —CH₂Cl, —CH₂CN, and —CH₂NO₂.

In certain embodiments, Y is a saturated 3-6 membered monocyclic ringhaving 0-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur, wherein Y is substituted with 1-4 R^(e) groups, wherein eachR^(e) is as defined above and described herein.

In some embodiments, Y is a saturated 3-4 membered heterocyclic ringhaving 1 heteroatom selected from oxygen or nitrogen wherein said ringis substituted with 1-2 R^(e) groups, wherein each R^(e) is as definedabove and described herein. Exemplary such rings are epoxide and oxetanerings, wherein each ring is substituted with 1-2 R^(e) groups, whereineach R^(e) is as defined above and described herein.

In other embodiments, Y is a saturated 5-6 membered heterocyclic ringhaving 1-2 heteroatom selected from oxygen or nitrogen wherein said ringis substituted with 1-4 R^(e) groups, wherein each R^(e) is as definedabove and described herein. Such rings include piperidine andpyrrolidine, wherein each ring is substituted with 1-4 R^(e) groups,wherein each R^(e) is as defined above and described herein. In certainembodiments, Y is

wherein each R, Q, Z, and R^(e) is as defined above and describedherein.

In some embodiments, Y is a saturated 3-6 membered carbocyclic ring,wherein said ring is substituted with 1-4 R^(e) groups, wherein eachR^(e) is as defined above and described herein. In certain embodiments,Y is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, wherein eachring is substituted with 1-4 R^(e) groups, wherein each R^(e) is asdefined above and described herein. In certain embodiments, Y is

wherein R^(e) is as defined above and described herein. In certainembodiments, Y is cyclopropyl optionally substituted with halogen, CN orNO₂.

In certain embodiments, Y is a partially unsaturated 3-6 memberedmonocyclic ring having 0-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, wherein said ring is substituted with 1-4R^(e) groups, wherein each R^(e) is as defined above and describedherein.

In some embodiments, Y is a partially unsaturated 3-6 memberedcarbocyclic ring, wherein said ring is substituted with 1-4 R^(e)groups, wherein each R^(e) is as defined above and described herein. Insome embodiments, Y is cyclopropenyl, cyclobutenyl, cyclopentenyl, orcyclohexenyl wherein each ring is substituted with 1-4 R^(e) groups,wherein each R^(e) is as defined above and described herein. In certainembodiments, Y is

wherein each R^(e) is as defined above and described herein.

In certain embodiments, Y is a partially unsaturated 4-6 memberedheterocyclic ring having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, wherein said ring is substituted with 1-4R^(e) groups, wherein each R^(e) is as defined above and describedherein. In certain embodiments, Y is selected from:

wherein each R and R^(e) is as defined above and described herein.

In certain embodiments, Y is a 6-membered aromatic ring having 0-2nitrogens wherein said ring is substituted with 1-4 R^(e) groups,wherein each R^(e) group is as defined above and described herein. Incertain embodiments, Y is phenyl, pyridyl, or pyrimidinyl, wherein eachring is substituted with 1-4 R^(e) groups, wherein each R^(e) is asdefined above and described herein.

In some embodiments, Y is selected from:

wherein each R^(e) is as defined above and described herein.

In other embodiments, Y is a 5-membered heteroaryl ring having 1-3heteroatoms independently selected from nitrogen, oxygen, or sulfur,wherein said ring is substituted with 1-3 R^(e) groups, wherein eachR^(e) group is as defined above and described herein. In someembodiments, Y is a 5 membered partially unsaturated or aryl ring having1-3 heteroatoms independently selected from nitrogen, oxygen, andsulfur, wherein said ring is substituted with 1-4 R^(e) groups, whereineach R^(e) group is as defined above and described herein. Exemplarysuch rings are isoxazolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl,pyrrolyl, furanyl, thienyl, triazole, thiadiazole, and oxadiazole,wherein each ring is substituted with 1-3 R^(e) groups, wherein eachR^(e) group is as defined above and described herein. In certainembodiments, Y is selected from:

wherein each R and R^(e) is as defined above and described herein.

In certain embodiments, Y is an 8-10 membered bicyclic, saturated,partially unsaturated, or aryl ring having 0-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, wherein said ring issubstituted with 1-4 R^(e) groups, wherein R^(e) is as defined above anddescribed herein. According to another aspect, Y is a 9-10 memberedbicyclic, partially unsaturated, or aryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, wherein saidring is substituted with 1-4 R^(e) groups, wherein R^(e) is as definedabove and described herein. Exemplary such bicyclic rings include2,3-dihydrobenzo[d]isothiazole, wherein said ring is substituted with1-4 R^(e) groups, wherein R^(e) is as defined above and describedherein.

As defined generally above, each R^(e) group is independently selectedfrom -Q-Z, oxo, NO₂, halogen, CN, a suitable leaving group, or C₁₋₆aliphatic optionally substituted with oxo, halogen, NO₂, or CN, whereinQ is a covalent bond or a bivalent C₁₋₆ saturated or unsaturated,straight or branched, hydrocarbon chain, wherein one or two methyleneunits of Q are optionally and independently replaced by —N(R)—, —S—,—O—, —C(O)—, —OC(O)—, —C(O)O—, —SO—, or —SO₂—, —N(R)C(O)—, —C(O)N(R)—,—N(R)SO₂—, or —SO₂N(R)—; and Z is hydrogen or C₁₋₆ aliphatic optionallysubstituted with oxo, halogen, NO₂, or CN.

In certain embodiments, R^(e) is C₁₋₆ aliphatic optionally substitutedwith oxo, halogen, NO₂, or CN. In other embodiments, R^(e) is oxo, NO₂,halogen, or CN.

In some embodiments, R^(e) is -Q-Z, wherein Q is a covalent bond and Zis hydrogen (i.e., R^(e) is hydrogen). In other embodiments, R^(e) is-Q-Z, wherein Q is a bivalent C₁₋₆ saturated or unsaturated, straight orbranched, hydrocarbon chain, wherein one or two methylene units of Q areoptionally and independently replaced by —NR—, —NRC(O)—, —C(O)NR—, —S—,—O—, —C(O)—, —SO—, or —SO₂—. In other embodiments, Q is a bivalent C₂₋₆straight or branched, hydrocarbon chain having at least one double bond,wherein one or two methylene units of Q are optionally and independentlyreplaced by —NR—, —NRC(O)—, —C(O)NR—, —S—, —O—, —C(O)—, —SO—, or —SO₂—.In certain embodiments, the Z moiety of the R^(e) group is hydrogen. Insome embodiments, -Q-Z is —NHC(O)CH═CH₂ or —C(O)CH═CH₂.

In certain embodiments, each R^(e) is independently selected from fromoxo, NO₂, CN, fluoro, chloro, —NHC(O)CH═CH₂, —C(O)CH═CH₂, —CH₂CH═CH₂,—C≡CH, —C(O)OCH₂Cl, —C(O)OCH₂F, —C(O)OCH₂CN, —C(O)CH₂Cl, —C(O)CH₂F,—C(O)CH₂CN, or —CH₂C(O)CH₃.

In certain embodiments, R^(e) is a suitable leaving group, ie a groupthat is subject to nucleophilic displacement. A “suitable leaving” is achemical group that is readily displaced by a desired incoming chemicalmoiety such as the thiol moiety of a cysteine of interest. Suitableleaving groups are well known in the art, e.g., see, “Advanced OrganicChemistry,” Jerry March, 5^(th) Ed., pp. 351-357, John Wiley and Sons,N.Y. Such leaving groups include, but are not limited to, halogen,alkoxy, sulphonyloxy, optionally substituted alkylsulphonyloxy,optionally substituted alkenylsulfonyloxy, optionally substitutedarylsulfonyloxy, acyl, and diazonium moieties. Examples of suitableleaving groups include chloro, iodo, bromo, fluoro, acetoxy,methanesulfonyloxy (mesyloxy), tosyloxy, triflyloxy,nitro-phenylsulfonyloxy (nosyloxy), and bromo-phenylsulfonyloxy(brosyloxy).

In certain embodiments, the following embodiments and combinations of-L-Y apply:

-   -   (a) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and one or two additional        methylene units of L are optionally and independently replaced        by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—,        —OC(O)—, —C(O)O—, cyclopropylene, —O—, —N(R)—, or —C(O)—; and Y        is hydrogen or C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN; or    -   (b) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —C(O)—, —NRC(O)—, —C(O)NR—,        —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—,        and one or two additional methylene units of L are optionally        and independently replaced by cyclopropylene, —O—, —N(R)—, or        —C(O)—; and Y is hydrogen or C₁₋₆ aliphatic optionally        substituted with oxo, halogen, NO₂, or CN; or    -   (c) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —C(O)—, and one or two        additional methylene units of L are optionally and independently        replaced by cyclopropylene, —O—, —N(R)—, or —C(O)—; and Y is        hydrogen or C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN; or    -   (d) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —C(O)—; and Y is hydrogen or        C₁₋₆ aliphatic optionally substituted with oxo, halogen, NO₂, or        CN; or    -   (e) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —OC(O)—; and Y is hydrogen or        C₁₋₆ aliphatic optionally substituted with oxo, halogen, NO₂, or        CN; or    -   (f) L is —NRC(O)CH═CH—, —NRC(O)CH═CHCH₂N(CH₃)—,        —NRC(O)CH═CHCH₂O—, —CH₂NRC(O)CH═CH—, —NRSO₂CH═CH—,        —NRSO₂CH═CHCH₂—, —NRC(O)(C═N₂)—, —NRC(O)(C═N₂)C(O)—,        —NRC(O)CH═CHCH₂O—, —NRC(O)C(═CH₂)CH₂—, —CH₂NRC(O)—,        —CH₂CH₂NRC(O)—, or —CH₂NRC(O)cyclopropylene-; wherein R is H or        optionally substituted C₁₋₆ aliphatic; and Y is hydrogen or C₁₋₆        aliphatic optionally substituted with oxo, halogen, NO₂, or CN;        or    -   (g) L is —NHC(O)CH═CH—, —NHC(O)CH═CHCH₂N(CH₃)—,        —NHC(O)CH═CHCH₂O—, —CH₂NHC(O)CH═CH—, —NHSO₂CH═CH—,        —NHSO₂CH═CHCH₂—, —NHC(O)(C═N₂)—, —NHC(O)(C═N₂)C(O)—,        —NHC(O)C(═CH₂)CH₂—, —CH₂NHC(O)—, —CH₂CH₂NHC(O)—, or        —CH₂NHC(O)cyclopropylene-; and Y is hydrogen or C₁₋₆ aliphatic        optionally substituted with oxo, halogen, NO₂, or CN; or    -   (h) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one alkylidenyl double bond and at least        one methylene unit of L is replaced by —C(O)—, —NRC(O)—,        —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or        —C(O)O—, and one or two additional methylene units of L are        optionally and independently replaced by cyclopropylene, —O—,        —N(R)—, or —C(O)—; and Y is hydrogen or C₁₋₆ aliphatic        optionally substituted with oxo, halogen, NO₂, or CN; or    -   (i) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one triple bond and one or two additional        methylene units of L are optionally and independently replaced        by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—,        —OC(O)—, or —C(O)O—, and Y is hydrogen or C₁₋₆ aliphatic        optionally substituted with oxo, halogen, NO₂, or CN; or    -   (j) L is —C≡C—, —C≡CCH₂N(isopropyl)-, —NHC(O)C≡CCH₂CH₂—,        —CH₂—C≡C—CH₂—, —C≡CCH₂O—, —CH₂C(O)C≡C—, —C(O)C≡C—, or        —CH₂OC(≡O)C≡C—; and Y is hydrogen or C₁₋₆ aliphatic optionally        substituted with oxo, halogen, NO₂, or CN; or    -   (k) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein one methylene unit of L is replaced by cyclopropylene        and one or two additional methylene units of L are independently        replaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—,        —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—; and Y is hydrogen or C₁₋₆        aliphatic optionally substituted with oxo, halogen, NO₂, or CN;        or    -   (l) Lisa covalent bond and Y is selected from:        -   (i) C₁-6 alkyl substituted with oxo, halogen, NO₂, or CN;        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or ‘ ’        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or

-   -    wherein each R^(e) is as defined above and described herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or

-   -   -   -   wherein each R and R^(e) is as defined above and                described herein; or

        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or

-   -   -   -   wherein each R^(e) is as defined above and described                herein; or

        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or

-   -   -   -   wherein each R and R^(e) is as defined above and                described herein; or

        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (m) L is —C(O)— and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN; or        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or

-   -   -   -   wherein each R and R^(e) is as defined above and                described herein; or

        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or

-   -   -   -   wherein each R^(e) is as defined above and described                herein; or

        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or

-   -   -   -   wherein each R and R^(e) is as defined above and                described herein; or

        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (n) L is —N(R)C(O)— and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN; or        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or

-   -   -   -   wherein each R and R^(e) is as defined above and                described herein; or

        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or

-   -   -   -   wherein each R^(e) is as defined above and described                herein; or

        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or

-   -   -   -   wherein each R and R^(e) is as defined above and                described herein; or

        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (o) L is a bivalent C₁₋₈ saturated or unsaturated, straight or        branched, hydrocarbon chain; and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN;        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or

-   -   -   -   wherein each R and R^(e) is as defined above and                described herein; or

        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or

-   -   -   -   wherein each R^(e) is as defined above and described                herein; or

        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or

-   -   -   -   wherein each R and R^(e) is as defined above and                described herein; or

        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (p) L is a covalent bond, —CH₂—, —NH—, —C(O)—, —CH₂NH—, —NHCH₂—,        —NHC(O)—, —NHC(O)CH₂OC(O)—, —CH₂NHC(O)—, —NHSO₂—, —NHSO₂CH₂—,        —NHC(O)CH₂OC(O)—, or —SO₂NH—; and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN; or        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or

-   -   -   -   wherein each R and R^(e) is as defined above and                described herein; or

        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein;

-   -   -   -   wherein each R^(e) is as defined above and described                herein; or

        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or

-   -   -   -   wherein each R and R^(e) is as defined above and                described herein; or

        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein.

    -   (q) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein two or three methylene units of L are optionally and        independently replaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—,        —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, —C(O)O—, cyclopropylene,        —O—, —N(R)—, or —C(O)—; and Y is hydrogen or C₁₋₆ aliphatic        optionally substituted with oxo, halogen, NO₂, or CN.

    -   (r) L-Y is “pro-warhead” that is converted in vitro or in vivo        to an irreversible warhead. In certain embodiments, L-Y is

wherein LG is a leaving group. In certain embodiments, L-Y is

In certain embodiments, the “pro-warhead” is converted to anirreversible warhead according to the following:

In certain embodiments, the Y group of any of the formulae herein isselected from those set forth in Table 1, wherein each wavy lineindicates the point of attachment to the rest of the molecule.

In certain embodiments, R¹ is -L-Y, wherein:

-   L is a covalent bond or a bivalent C₁₋₈ saturated or unsaturated,    straight or branched, hydrocarbon chain, wherein one, two, or three    methylene units of L are optionally and independently replaced by    —N(R)—, —N(R)C(O)—, —N(R)SO₂—, —O—, —C(O)—, or —SO₂—; and-   Y is hydrogen, or C₁₋₆ aliphatic optionally substituted with oxo,    halogen, N(R)₂, NO₂, or CN.

In certain embodiments, the Y group of R¹ group, -L-Y, is selected fromthose set forth in Table 3, below, wherein each wavy line indicates thepoint of attachment to the rest of the molecule.

TABLE 3 Exemplary Y groups:

wherein each R^(e) is independently a suitable leaving group, NO₂, CN,or oxo.

In certain embodiments, R¹ is —C(O)CH₂CH₂C(O)CH═C(CH₃)₂,—C(O)CH₂CH₂C(O)CH═CH(cyclopropyl), —C(O)CH₂CH₂C(O)CH═CHCH₃,C(O)CH₂CH₂C(O)CH═CHCH₂CH₃, or —C(O)CH₂CH₂C(O)C(═CH₂)CH₃. In certainembodiments, R¹ is —C(O)CH₂NHC(O)CH═CH₂,—C(O)CH₂NHC(O)CH₂CH₂C(O)CH═CHCH₃, or —C(O)CH₂NHC(O)CH₂CH₂C(O)C(═CH₂)CH₃.In certain embodiments, R¹ is —S(O)₂CH₂CH₂NHC(O)CH₂CH₂C(O)CH═C(CH₃)₂,S(O)₂CH₂CH₂NHC(O)CH₂CH₂C(O)CH═CHCH₃, orS(O)₂CH₂CH₂NHC(O)CH₂CH₂C(O)CH═CH₂. In certain embodiments, R¹ is—C(O)(CH₂)₃NHC(O)CH₂CH₂C(O)CH═CHCH₃ or—C(O)(CH₂)₃NHC(O)CH₂CH₂C(O)CH═CH₂.

In certain embodiments, R¹ is —C≡CH, —C≡CCH₂NH(isopropyl),—NHC(O)C≡CCH₂CH₃, —CH₂—C≡C—CH₃, —C≡CCH₂OH, —CH₂C(O)C≡CH, —C(O)C≡CH, or—CH₂OC(═O)C≡CH. In some embodiments, R¹ is selected from —NHC(O)CH═CH₂,—NHC(O)CH═CHCH₂N(CH₃)₂, or —CH₂NHC(O)CH═CH₂.

In certain embodiments, R¹ is selected from those set forth in Table 4,below, wherein each wavy line indicates the point of attachment to therest of the molecule.

TABLE 4 Exemplary R¹ Groups

wherein each R^(e) is independently a suitable leaving group, NO₂, CN,or oxo.

In certain embodiments, R¹ is selected from

In certain embodiments, R¹ is selected from

In certain embodiments, R¹ is selected from:

In some embodiments, R¹ is selected from those depicted in Table 1 andTable 2, above.

As defined generally above, R¹ is a warhead group. Without wishing to bebound by any particular theory, it is believed that such R¹ groups, i.e.warhead groups, are particularly suitable for covalently binding to akey cysteine residue in the binding domain of certain protein kinases.Protein kinases having a cysteine residue in the binding domain areknown to one of ordinary skill in the art and include FGFR4, or a mutantthereof. FIG. 4 provides SEQ ID NO. 1, which is the amino acid sequenceof FGFR4. In certain embodiments, compounds of the present inventionhave a warhead group characterized in that inventive compounds targetthe cysteine 552 residue (which is highlighted by a box in FIG. 4 and inbold with underlining below):

-   -   FGFR4 subsequence containing C552:    -   LGVCTQEGPLYVIVECAAKGNLREFLRARRP

Thus, in some embodiments, R¹ is characterized in that the -L-Y moietyis capable of covalently binding to the cysteine 552 residue therebyirreversibly inhibiting the enzyme. The cysteine residue is Cys552 ofFGFR4, or a mutant thereof, where the provided residue numbering is inaccordance with Uniprot P22455).

One of ordinary skill in the art will recognize that a variety ofwarhead groups, as defined herein, are suitable for such covalentbonding.

In certain embodiments, R¹ is characterized in that the -L-Y moiety iscapable of covalently binding to a cysteine residue of FGFR4, therebyirreversibly inhibiting the enzyme. In some embodiments, the cysteineresidue is Cys 552.

One of ordinary skill in the art will recognize that a variety ofwarhead groups, as defined herein, are suitable for such covalentbonding. Such R¹ groups include, but are not limited to, those describedherein and depicted above.

In certain embodiments, the present invention provides any compounddepicted in any table above, or a pharmaceutically acceptable saltthereof.

As described herein, compounds of the present invention are irreversibleinhibitors of FGFR4, or a mutant thereof.

In certain embodiments, the present invention provides a conjugate ofthe formula A:

Cys552-modifier-inhibitor moiety  A

wherein:

the Cys552 is Cys552 of FGFR4;

the modifier is a bivalent group resulting from covalent bonding of awarhead group with the Cys552 of FGFR4 kinase;the warhead group is a functional group capable of covalently binding toCys552; andthe inhibitor moiety is a moiety that binds in the binding site of theFGFR4 kinase.

In certain embodiments, the inhibitor moiety of conjugate A is offormula I-i:

wherein the wavy bond indicates the point of attachment to Cys552 ofconjugate A via the modifier; and wherein each of Ring A, R², R³, X¹,X², X³, X⁴, X⁵, Y, G, T, and q, of formula I-i is as defined for formulaI above and as defined and described in embodiments herein.

In certain embodiments, the inhibitor moiety of conjugate A is offormula I-a-i:

wherein the wavy bond indicates the point of attachment to Cys552 ofconjugate A via the modifier; and wherein each of Ring A, R², R³, R⁴,R⁵, R^(5′), R^(a), T, and q, of formula I-a-i is as defined for formulaI-a above and as defined and described in embodiments herein.

In certain embodiments, the inhibitor moiety of conjugate A is offormula I-b-i:

wherein the wavy bond indicates the point of attachment to Cys552 ofconjugate A via the modifier; and wherein each of Ring A, R², R³, R⁴, T,and q, of formula I-b-i is as defined for formula I-b above and asdefined and described in embodiments herein.

In certain embodiments, the inhibitor moiety of conjugate A is offormula I-c-i:

wherein the wavy bond indicates the point of attachment to Cys552 ofconjugate A via the modifier; and wherein each of R², R³, R⁴, R^(a), andq, of formula I-c-i is as defined for formula I-c above and as definedand described in embodiments herein.

In certain embodiments, the inhibitor moiety of conjugate A is offormula I-d-i:

wherein the wavy bond indicates the point of attachment to Cys552 ofconjugate A via the modifier; and wherein each of R², R³, R⁴, R^(a), andq, of formula I-d-i is as defined for formula I-d above and as definedand described in embodiments herein.

In certain embodiments, the inhibitor moiety of conjugate A is offormula I-e-i:

wherein the wavy bond indicates the point of attachment to Cys552 ofconjugate A via the modifier; and wherein each of Ring A, R², R³, R⁵,R^(5′), R^(a), and q, of formula I-e-i is as defined for formula I-eabove and as defined and described in embodiments herein.

In certain embodiments, the inhibitor moiety of conjugate A is offormula I-f-i:

wherein the wavy bond indicates the point of attachment to Cys552 ofconjugate A via the modifier; and wherein each of Ring A, R², R³, R⁵,R^(5′), R^(a), and q, of formula I-f-i is as defined for formula I-fabove and as defined and described in embodiments herein.

In certain embodiments, the inhibitor moiety of conjugate A is offormula I-g-i:

wherein the wavy bond indicates the point of attachment to Cys552 ofconjugate A via the modifier; and wherein each of Ring A, R², R³, R⁴,R^(a), and q, of formula I-g-i is as defined for formula I-g above andas defined and described in embodiments herein.

In certain embodiments, the inhibitor moiety of conjugate A is offormula I-h-i:

wherein the wavy bond indicates the point of attachment to Cys552 ofconjugate A via the modifier; and wherein each of Ring A, R², R³, R⁴,R^(a), and q, of formula I-h-i is as defined for formula I-h above andas defined and described in embodiments herein.

In certain embodiments, the inhibitor moiety of conjugate A is offormula I-j-i:

wherein the wavy bond indicates the point of attachment to Cys552 ofconjugate A via the modifier; and wherein each of Ring A, R², R⁴, R^(a),and q, of formula I-j-i is as defined for formula I-j above and asdefined and described in embodiments herein.

In certain embodiments, the inhibitor moiety of conjugate A is offormula I-k-i:

wherein the wavy bond indicates the point of attachment to Cys552 ofconjugate A via the modifier; and wherein each of Ring A, R², R³, R⁴,R^(a), and q, of formula I-k-i is as defined for formula I-k above andas defined and described in embodiments herein.

In certain embodiments, the inhibitor moiety of conjugate A is offormula I-n-i:

wherein the wavy bond indicates the point of attachment to Cys552 ofconjugate A via the modifier; and wherein each of Ring A, R², R³, R⁴,and q, of formula I-n-i is as defined for formula I-n above and asdefined and described in embodiments herein.

In certain embodiments, the inhibitor moiety of conjugate A is offormula I-q-i:

wherein the wavy bond indicates the point of attachment to Cys552 ofconjugate A via the modifier; and wherein each of Ring A, R², R³, R⁴,R^(a), T, and q, of formula I-q-i is as defined for formula I-q aboveand as defined and described in embodiments herein.

In certain embodiments, the present invention provides a conjugate ofany of the formulae below:

wherein each of Cys552, Modifier, Ring A, R², R³, R⁴, R⁵, R^(5′), R^(a),X¹, X², X³, X⁴, X⁵, T, Y, G and q, with respect to the above formulae isas defined and described in embodiments herein for formulae I, I-a, I-b,I-c, I-d, I-e, I-f, I-g, I-h, I-j, I-k, I-n, and I-q.

In other embodiments, the modifier moiety of any of conjugate describedabove is selected from those set forth in Table 5, below. Exemplarymodifiers further include any bivalent group resulting from covalentbonding of a warhead moiety found in Table 1, Table 2, or Table 4 withcysteine 552 of FGFR4. It will be understood that the exemplarymodifiers below are shown as conjugated to the sulfhydryl of Cys552.

TABLE 5 Exemplary Modifiers Conjugated to Cys552:

4. Uses, Formulation and Administration Pharmaceutically AcceptableCompositions

According to another embodiment, the invention provides a compositioncomprising a compound of this invention or a pharmaceutically acceptablederivative thereof and a pharmaceutically acceptable carrier, adjuvant,or vehicle. The amount of compound in compositions of this invention issuch that is effective to measurably inhibit a protein kinase,particularly FGFR4, or a mutant thereof, in a biological sample or in apatient. In certain embodiments, the amount of compound in compositionsof this invention is such that is effective to measurably inhibit FGFR4,or a mutant thereof, in a biological sample or in a patient. In certainembodiments, a composition of this invention is formulated foradministration to a patient in need of such composition. In someembodiments, a composition of this invention is formulated for oraladministration to a patient.

The term “patient”, as used herein, means an animal, preferably amammal, and most preferably a human.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat are used in the compositions of this invention include, but are notlimited to, ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

A “pharmaceutically acceptable derivative” means any non-toxic salt,ester, salt of an ester or other derivative of a compound of thisinvention that, upon administration to a recipient, is capable ofproviding, either directly or indirectly, a compound of this inventionor an inhibitorily active metabolite or residue thereof.

As used herein, the term “inhibitorily active metabolite or residuethereof” means that a metabolite or residue thereof is also an inhibitorof FGFR4, or a mutant thereof.

Compositions of the present invention are administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously. Sterile injectable forms of thecompositions of this invention include aqueous or oleaginous suspension.These suspensions are formulated according to techniques known in theart using suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation is also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that are employed are water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium.

For this purpose, any bland fixed oil employed includes synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions also contain a long-chain alcohol diluent or dispersant,such as carboxymethyl cellulose or similar dispersing agents that arecommonly used in the formulation of pharmaceutically acceptable dosageforms including emulsions and suspensions. Other commonly usedsurfactants, such as Tweens, Spans and other emulsifying agents orbioavailability enhancers which are commonly used in the manufacture ofpharmaceutically acceptable solid, liquid, or other dosage forms arealso be used for the purposes of formulation.

Pharmaceutically acceptable compositions of this invention are orallyadministered in any orally acceptable dosage form. Exemplary oral dosageforms are capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents are optionally also added.

Alternatively, pharmaceutically acceptable compositions of thisinvention are administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

Pharmaceutically acceptable compositions of this invention are alsoadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches are also used.

For topical applications, provided pharmaceutically acceptablecompositions are formulated in a suitable ointment containing the activecomponent suspended or dissolved in one or more carriers. Exemplarycarriers for topical administration of compounds of this aremineral oil,liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene compound, emulsifying wax and water. Alternatively,provided pharmaceutically acceptable compositions can be formulated in asuitable lotion or cream containing the active components suspended ordissolved in one or more pharmaceutically acceptable carriers. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, provided pharmaceutically acceptable compositionsare optionally formulated as micronized suspensions in isotonic, pHadjusted sterile saline, or, preferably, as solutions in isotonic, pHadjusted sterile saline, either with or without a preservative such asbenzylalkonium chloride. Alternatively, for ophthalmic uses, thepharmaceutically acceptable compositions are formulated in an ointmentsuch as petrolatum.

Pharmaceutically acceptable compositions of this invention areoptionally administered by nasal aerosol or inhalation. Suchcompositions are prepared according to techniques well-known in the artof pharmaceutical formulation and are prepared as solutions in saline,employing benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons, and/or otherconventional solubilizing or dispersing agents.

Most preferably, pharmaceutically acceptable compositions of thisinvention are formulated for oral administration. Such formulations maybe administered with or without food. In some embodiments,pharmaceutically acceptable compositions of this invention areadministered without food. In other embodiments, pharmaceuticallyacceptable compositions of this invention are administered with food.

The amount of compounds of the present invention that are optionallycombined with the carrier materials to produce a composition in a singledosage form will vary depending upon the host treated, the particularmode of administration. Preferably, provided compositions should beformulated so that a dosage of between 0.01-100 mg/kg body weight/day ofthe inhibitor can be administered to a patient receiving thesecompositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

Uses of Compounds and Pharmaceutically Acceptable Compositions

Compounds and compositions described herein are generally useful for theinhibition of protein kinase activity of one or more enzymes.

Drug resistance is emerging as a significant challenge for targetedtherapies. For example, drug resistance has been reported for Gleevec®and Iressa®, as well as several other kinase inhibitors in development.In addition, drug resistance has been reported for the cKit and PDGFRreceptors. It has been reported that irreversible inhibitors may beeffective against drug resistant forms of protein kinases (Kwak, E. L.,R. Sordella, et al. (2005). “Irreversible inhibitors of the EGF receptormay circumvent acquired resistance to gefitinib.” PNAS 102(21):7665-7670.) Without wishing to be bound by any particular theory, thecompounds of the present invention are effective inhibitors of drugresistant forms of protein kinases.

Examples of kinases that are inhibited by the compounds and compositionsdescribed herein and against which the methods described herein areuseful include FGFR4, or a mutant thereof. In some embodiments, aprovided compound inhibits FGFR4 selectively as compared to other FGFRkinases.

The activity of a compound utilized in this invention as a test compoundof FGFR4, or a mutant thereof, may be assayed in vitro, in vivo or in acell line. In vitro assays include assays that determine inhibition ofeither the phosphorylation activity and/or the subsequent functionalconsequences, or ATPase activity of activated FGFR4, or a mutantthereof. Alternate in vitro assays quantitate the ability of the testcompound to bind to FGFR4. Inhibitor binding may be measured byradiolabeling the inhibitor prior to binding, isolating the testcompound /FGFR4 complex and determining the amount of radiolabel bound.Alternatively, test compound binding may be determined by running acompetition experiment where new test compounds are incubated with FGFR4bound to known radioligands. Detailed conditions for assaying a compoundutilized in this invention as a test compound of FGFR4, or a mutantthereof, are set forth in the Examples below.

Protein tyrosine kinases are a class of enzymes that catalyze thetransfer of a phosphate group from ATP or GTP to a tyrosine residuelocated on a protein substrate. Receptor tyrosine kinases act totransmit signals from the outside of a cell to the inside by activatingsecondary messaging effectors via a phosphorylation event. A variety ofcellular processes are promoted by these signals, includingproliferation, carbohydrate utilization, protein synthesis,angiogenesis, cell growth, and cell survival.

(a) FGFR Family

The fibroblast growth factor (FGF) family of protein tyrosine kinase(PTK) receptors regulates a diverse array of physiologic functionsincluding mitogenesis, wound healing, cell differentiation andangiogenesis, and development. Both normal and malignant cell growth aswell as proliferation are affected by changes in local concentration ofFGFs, extracellular signalling molecules which act as autocrine as wellas paracrine factors. Autocrine FGF signalling may be particularlyimportant in the progression of steroid hormone-dependent cancers to ahormone independent state (Powers, et al. (2000) Endocr. Relat. Cancer,7, 165-197).

FGFs and their receptors are expressed at increased levels in severaltissues and cell lines and overexpression is believed to contribute tothe malignant phenotype.

The two prototypic members are acidic fibroblast growth factor (aFGF orFGF1) and basic fibroblast growth factor (bFGF or FGF2), and to date, atleast twenty distinct FGF family members have been identified. Thecellular response to FGFs is transmitted via four types of high affinitytransmembrane protein tyrosine-kinase fibroblast growth factor receptors(FGFR) numbered 1 to 4 (FGFR1 to FGFR4). Upon ligand binding, thereceptors dimerize and auto- or trans-phosphorylate specific cytoplasmictyrosine residues to transmit an intracellular signal that ultimatelyregulates nuclear transcription factor effectors.

Compounds which inhibit FGFR will be useful in providing a means ofpreventing the growth or inducing apoptosis in tumors, particularly byinhibiting angiogenesis. It is therefore anticipated that the compoundswill prove useful in treating or preventing proliferative disorders suchas cancers. In particular tumours with activating mutants of receptortyrosine kinases or upregulation of receptor tyrosine kinases may beparticularly sensitive to the inhibitors.

Various studies described targeting of either FGFR4 kinase activity orits ligand FGF 19 with an antibody antagonist inhibited proliferationand induced apoptosis in cell line models. Ho et al (2009) Journal ofHepatology, 50 showed that one third of patients with a commonpolymorphism in the FGFR4 gene expressed high levels of mRNA and thesetumours were associated with high secreted levels of the hepatocellularcarcinoma marker alpha-fetoprotein.

In certain embodiments, the invention provides a method for inhibitingFGFR4, or a mutant thereof, activity in a patient or in a biologicalsample comprising the step of administering to said patient orcontacting said biological sample with a compound according to theinvention.

In certain embodiments, the FGFR4, or a mutant thereof, activity isinhibited irreversibly. In certain embodiments, FGFR4, or a mutantthereof, activity is inhibited irreversibly by covalently modifying Cys552 of FGFR4.

In certain embodiments, the invention provides a method for treating aFGFR4-mediated disorder in a patient in need thereof, comprising thestep of administering to said patient a compound according to theinvention, or a pharmaceutically acceptable composition thereof.

In some embodiments, the present invention provides a method fortreating hepatocellular carcinoma in a patient in need thereof,comprising the step of administering to said patient a compoundaccording to the invention, or a pharmaceutically acceptable compositionthereof.

In some embodiments, the present invention provides a method fortreating Rhabdomyosarcoma, esophageal cancer, breast cancer, or cancerof a head or neck, in a patient in need thereof, comprising the step ofadministering to said patient a compound according to the invention, ora pharmaceutically acceptable composition thereof.

As used herein, the term “clinical drug resistance” refers to the lossof susceptibility of a drug target to drug treatment as a consequence ofmutations in the drug target.

As used herein, the term “resistance” refers to changes in the wild-typenucleic acid sequence coding a target protein, and/or the proteinsequence of the target, which changes decrease or abolish the inhibitoryeffect of the inhibitor on the target protein.

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment is administered afterone or more symptoms have developed. In other embodiments, treatment isadministered in the absence of symptoms. For example, treatment isadministered to a susceptible individual prior to the onset of symptoms(e.g., in light of a history of symptoms and/or in light of genetic orother susceptibility factors). Treatment is also continued aftersymptoms have resolved, for example to prevent or delay theirrecurrence.

The compounds and compositions, according to the method of the presentinvention, are administered using any amount and any route ofadministration effective for treating or lessening the severity of adisorder provided above. The exact amount required will vary fromsubject to subject, depending on the species, age, and general conditionof the subject, the severity of the infection, the particular agent, itsmode of administration, and the like. Compounds of the invention arepreferably formulated in dosage unit form for ease of administration anduniformity of dosage. The expression “dosage unit form” as used hereinrefers to a physically discrete unit of agent appropriate for thepatient to be treated. It will be understood, however, that the totaldaily usage of the compounds and compositions of the present inventionwill be decided by the attending physician within the scope of soundmedical judgment. The specific effective dose level for any particularpatient or organism will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts.

Pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention are administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms optionally contain inert diluents commonly usedin the art such as, for example, water or other solvents, solubilizingagents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions are formulated according to the known art usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation are also a sterile injectable solution,suspension or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This is accomplished by the useof a liquid suspension of crystalline or amorphous material with poorwater solubility. The rate of absorption of the compound then dependsupon its rate of dissolution that, in turn, may depend upon crystal sizeand crystalline form. Alternatively, delayed absorption of aparenterally administered compound form is accomplished by dissolving orsuspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form also optionally comprise buffering agents.

Solid compositions of a similar type are also employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype are also employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms optionally also comprisebuffering agents. They optionally contain opacifying agents and can alsobe of a composition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as required. Ophthalmicformulation, ear drops, and eye drops are also contemplated as beingwithin the scope of this invention. Additionally, the present inventioncontemplates the use of transdermal patches, which have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms can be made by dissolving or dispensing the compoundin the proper medium. Absorption enhancers can also be used to increasethe flux of the compound across the skin. The rate can be controlled byeither providing a rate controlling membrane or by dispersing thecompound in a polymer matrix or gel.

According to one embodiment, the invention relates to a method ofinhibiting protein kinase activity in a biological sample comprising thestep of contacting said biological sample with a compound of thisinvention, or a composition comprising said compound.

According to another embodiment, the invention relates to a method ofinhibiting FGFR4, or a mutant thereof, activity in a biological samplecomprising the step of contacting said biological sample with a compoundof this invention, or a composition comprising said compound. In certainembodiments, the invention relates to a method of irreversiblyinhibiting FGFR4, or a mutant thereof, activity in a biological samplecomprising the step of contacting said biological sample with a compoundof this invention, or a composition comprising said compound.

The term “biological sample”, as used herein, includes, withoutlimitation, cell cultures or extracts thereof; biopsied materialobtained from a mammal or extracts thereof; and blood, saliva, urine,feces, semen, tears, or other body fluids or extracts thereof.

Inhibition of FGFR4, or a mutant thereof, activity in a biologicalsample is useful for a variety of purposes that are known to one ofskill in the art. Examples of such purposes include, but are not limitedto, blood transfusion, organ transplantation, biological specimenstorage, and biological assays.

Another embodiment of the present invention relates to a method ofinhibiting protein kinase activity in a patient comprising the step ofadministering to said patient a compound of the present invention, or acomposition comprising said compound.

According to another embodiment, the invention relates to a method ofinhibiting FGFR4, or a mutant thereof, activity in a patient comprisingthe step of administering to said patient a compound of the presentinvention, or a composition comprising said compound. According tocertain embodiments, the invention relates to a method of irreversiblyinhibiting FGFR4, or a mutant thereof, activity in a patient comprisingthe step of administering to said patient a compound of the presentinvention, or a composition comprising said compound. In otherembodiments, the present invention provides a method for treating adisorder mediated by FGFR4, or a mutant thereof, in a patient in needthereof, comprising the step of administering to said patient a compoundaccording to the present invention or pharmaceutically acceptablecomposition thereof. Such disorders are described in detail herein.

The compounds of this invention, or pharmaceutical compositions thereof,are optionally incorporated into compositions for coating an implantablemedical device, such as prostheses, artificial valves, vascular grafts,stents and catheters. Vascular stents, for example, have been used toovercome restenosis (re-narrowing of the vessel wall after injury).However, patients using stents or other implantable devices risk clotformation or platelet activation. These unwanted effects are preventedor mitigated by pre-coating the device with a pharmaceuticallyacceptable composition comprising a kinase inhibitor. Implantabledevices coated with a compound of this invention are another embodimentof the present invention.

5. Probe Compounds

In certain aspects, a compound of the present invention is tethered to adetectable moiety to form a probe compound. In one aspect, a probecompound of the invention comprises an irreversible protein kinaseinhibitor of any formulae as described herein, a detectable moiety, anda tethering moiety that attaches the inhibitor to the detectable moiety.

In some embodiments, such probe compounds of the present inventioncomprise a provided compound of any formulae as described herein,tethered to a detectable moiety, R^(t), by a bivalent tethering moiety,-T¹-. The tethering moiety is attached to a compound of the inventionvia Ring A, Ring B, or R¹. One of ordinary skill in the art willappreciate that when a tethering moiety is attached to R¹, R¹ is abivalent warhead group denoted as R^(1′). In certain embodiments, aprovided probe compound is selected from any of formula I-t:

wherein each of Ring A, R¹, R², R³, X¹, X², X³, X⁴, X⁵, G, Y, T, and q,is as defined above, and described in classes and subclasses herein,R^(2′) is a bivalent R²; T¹ is a bivalent tethering moiety; and R^(t) isa detectable moiety.

In some embodiments, R^(t) is a detectable moiety selected from aprimary label or a secondary label. In certain embodiments, R^(t) is adetectable moiety selected from a fluorescent label (e.g., a fluorescentdye or a fluorophore), a mass-tag, a chemiluminescent group, achromophore, an electron dense group, or an energy transfer agent. Insome embodiments, R^(t) is biotin, biotin sulfoxide, a radioisotope, ora fluorescent label.

As used herein, the term “detectable moiety” is used interchangeablywith the term “label” and “reporter” and relates to any moiety capableof being detected, e.g., primary labels and secondary labels. A presenceof a detectable moiety can be measured using methods for quantifying (inabsolute, approximate or relative terms) the detectable moiety in asystem under study. In some embodiments, such methods are well known toone of ordinary skill in the art and include any methods that quantify areporter moiety (e.g., a label, a dye, a photocrosslinker, a cytotoxiccompound, a drug, an affinity label, a photoaffinity label, a reactivecompound, an antibody or antibody fragment, a biomaterial, ananoparticle, a spin label, a fluorophore, a metal-containing moiety, aradioactive moiety, quantum dot(s), a novel functional group, a groupthat covalently or noncovalently interacts with other molecules, aphotocaged moiety, an actinic radiation excitable moiety, a ligand, aphotoisomerizable moiety, biotin, a biotin analog (e.g., biotinsulfoxide), a moiety incorporating a heavy atom, a chemically cleavablegroup, a photocleavable group, a redox-active agent, an isotopicallylabeled moiety, a biophysical probe, a phosphorescent group, achemiluminescent group, an electron dense group, a magnetic group, anintercalating group, a chromophore, an energy transfer agent, abiologically active agent, a detectable label, and any combination ofthe above).

Primary labels, such as radioisotopes (e.g., tritium, ³²P, ³³P, ³⁵S,¹⁴C, ¹²³I, ¹²⁴I, ¹²⁵I, or ¹³¹I), mass-tags are stable isotopes (e.g.,¹³C, ²H, ¹⁷O, ¹⁸O, ¹⁵N, ¹⁹F, and ¹²⁷I), positron emitting isotopes(e.g., ¹¹C, ¹⁸F, ¹³N, ¹²⁴I, and ¹⁵O), and fluorescent labels, which aresignal generating reporter groups which can be detected without furthermodifications. Detectable moities are analyzed by methods. Exemplarymethods are fluorescence, positron emission tomography, SPECT medicalimaging, chemiluminescence, electron-spin resonance, ultraviolet/visibleabsorbance spectroscopy, mass spectrometry, nuclear magnetic resonance,magnetic resonance, flow cytometry, autoradiography, scintillationcounting, phosphoimaging, and electrochemical methods.

The term “secondary label” as used herein refers to moieties such asbiotin and various protein antigens that require the presence of asecond intermediate for production of a detectable signal. For biotin,the secondary intermediate includes streptavidin-enzyme conjugates. Forantigen labels, secondary intermediates include antibody-enzymeconjugates. Some fluorescent groups act as secondary labels because theytransfer energy to another group in the process of nonradiativefluorescent resonance energy transfer (FRET), and the second groupproduces the detected signal.

The terms “fluorescent label”, “fluorescent dye”, and “fluorophore” asused herein refer to moieties that absorb light energy at a definedexcitation wavelength and emit light energy at a different wavelength.Examples of fluorescent labels include, but are not limited to: AlexaFluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, AlexaFluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, AlexaFluor 660 and Alexa Fluor 680), AMCA, AMCA-S, BODIPY dyes (BODIPY FL,BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 493/503, BODIPY 530/550,BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY630/650, BODIPY 650/665), Carboxyrhodamine 6G, carboxy-X-rhodamine(ROX), Cascade Blue, Cascade Yellow, Coumarin 343, Cyanine dyes (Cy3,Cy5, Cy3.5, Cy5.5), Dansyl, Dapoxyl, Dialkylaminocoumarin,4′,5′-Dichloro-2′,7′-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin,Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800),JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin,Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, RhodamineGreen, Rhodamine Red, Rhodol Green,2′,4′,5′,7′-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR),Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X,5(6)-Carboxyfluorescein, 2,7-Dichlorofluorescein,N,N-Bis(2,4,6-trimethylphenyl)-3,4:9,10-perylenebis(dicarboximide, HPTS,Ethyl Eosin, DY-490XL MegaStokes, DY-485XL MegaStokes, Adirondack Green520, ATTO 465, ATTO 488, ATTO 495, YOYO-1,5-FAM, BCECF,dichlorofluorescein, rhodamine 110, rhodamine 123, YO—PRO-1, SYTOXGreen, Sodium Green, SYBR Green I, Alexa Fluor 500, FITC, Fluo-3,Fluo-4, fluoro-emerald, YoYo-1 ssDNA, YoYo-1 dsDNA, YoYo-1, SYTORNASelect, Diversa Green-FP, Dragon Green, EvaGreen, Surf Green EX,Spectrum Green, NeuroTrace 500525, NBD-X, MitoTracker Green FM,LysoTracker Green DND-26, CBQCA, PA-GFP (post-activation), WEGFP(post-activation), F1ASH-CCXXCC, Azami Green monomeric, Azami Green,green fluorescent protein (GFP), EGFP (Campbell Tsien 2003), EGFP(Patterson 2001), Kaede Green,7-Benzylamino-4-Nitrobenz-2-Oxa-1,3-Diazole, Bexl, Doxorubicin, LumioGreen, and SuperGlo GFP.

The term “mass-tag” as used herein refers to any moiety that is capableof being uniquely detected by virtue of its mass using mass spectrometry(MS) detection techniques. Examples of mass-tags include electrophorerelease tags such asN-[3-[4′-[(p-Methoxytetrafluorobenzyl)oxy]phenyl]-3-methylglyceronyl]isonipecoticAcid, 4′-[2,3,5,6-Tetrafluoro-4-(pentafluorophenoxyl)]methylacetophenone, and their derivatives. The synthesis and utility of thesemass-tags is described in U.S. Pat. Nos. 4,650,750, 4,709,016,5,360,8191, 5,516,931, 5,602,273, 5,604,104, 5,610,020, and 5,650,270.Other examples of mass-tags include, but are not limited to,nucleotides, dideoxynucleotides, oligonucleotides of varying length andbase composition, oligopeptides, oligosaccharides, and other syntheticpolymers of varying length and monomer composition. A large variety oforganic molecules, both neutral and charged (biomolecules or syntheticcompounds) of an appropriate mass range (100-2000 Daltons) are also usedas mass-tags. Stable isotopes (e.g., ¹³C, ²H, ¹⁷O, ¹⁸O, and ¹⁵N) arealso used as mass-tags.

The term “chemiluminescent group,” as used herein, refers to a groupwhich emits light as a result of a chemical reaction without theaddition of heat. By way of example, luminol(5-amino-2,3-dihydro-1,4-phthalazinedione) reacts with oxidants likehydrogen peroxide (H₂O₂) in the presence of a base and a metal catalystto produce an excited state product (3-aminophthalate, 3-APA).

The term “chromophore,” as used herein, refers to a molecule whichabsorbs light of visible wavelengths, UV wavelengths or IR wavelengths.

The term “dye,” as used herein, refers to a soluble, coloring substancewhich contains a chromophore.

The term “electron dense group,” as used herein, refers to a group whichscatters electrons when irradiated with an electron beam. Such groupsinclude, but are not limited to, ammonium molybdate, bismuth subnitrate,cadmium iodide, carbohydrazide, ferric chloride hexahydrate,hexamethylene tetramine, indium trichloride anhydrous, lanthanumnitrate, lead acetate trihydrate, lead citrate trihydrate, lead nitrate,periodic acid, phosphomolybdic acid, phosphotungstic acid, potassiumferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate,silver proteinate (Ag Assay: 8.0-8.5%) “Strong”, silvertetraphenylporphin (S-TPPS), sodium chloroaurate, sodium tungstate,thallium nitrate, thiosemicarbazide (TSC), uranyl acetate, uranylnitrate, and vanadyl sulfate.

The term “energy transfer agent,” as used herein, refers to a moleculewhich either donates or accepts energy from another molecule. By way ofexample only, fluorescence resonance energy transfer (FRET) is adipole-dipole coupling process by which the excited-state energy of afluorescence donor molecule is non-radiatively transferred to anunexcited acceptor molecule which then fluorescently emits the donatedenergy at a longer wavelength.

The term “moiety incorporating a heavy atom,” as used herein, refers toa group which incorporates an ion of atom which is usually heavier thancarbon. In some embodiments, such ions or atoms include, but are notlimited to, silicon, tungsten, gold, lead, and uranium.

The term “photoaffinity label,” as used herein, refers to a label with agroup, which, upon exposure to light, forms a linkage with a moleculefor which the label has an affinity.

The term “photocaged moiety,” as used herein, refers to a group which,upon illumination at certain wavelengths, covalently or non-covalentlybinds other ions or molecules.

The term “photoisomerizable moiety,” as used herein, refers to a groupwherein upon illumination with light changes from one isomeric form toanother.

The term “radioactive moiety,” as used herein, refers to a group whosenuclei spontaneously give off nuclear radiation, such as alpha, beta, orgamma particles; wherein, alpha particles are helium nuclei, betaparticles are electrons, and gamma particles are high energy photons.

The term “spin label,” as used herein, refers to molecules which containan atom or a group of atoms exhibiting an unpaired electron spin (i.e. astable paramagnetic group) that in some embodiments are detected byelectron spin resonance spectroscopy and in other embodiments areattached to another molecule. Such spin-label molecules include, but arenot limited to, nitryl radicals and nitroxides, and in some embodimentsare single spin-labels or double spin-labels.

The term “quantum dots,” as used herein, refers to colloidalsemiconductor nanocrystals that in some embodiments are detected in thenear-infrared and have extremely high quantum yields (i.e., very brightupon modest illumination).

One of ordinary skill in the art will recognize that a detectable moietyis attached to a provided compound via a suitable substituent. As usedherein, the term “suitable substituent” refers to a moiety that iscapable of covalent attachment to a detectable moiety. Such moieties arewell known to one of ordinary skill in the art and include groupscontaining, e.g., a carboxylate moiety, an amino moiety, a thiol moiety,or a hydroxyl moiety, to name but a few. It will be appreciated thatsuch moieties are directly attached to a provided compound or via atethering moiety, such as a bivalent saturated or unsaturatedhydrocarbon chain.

In some embodiments, detectable moieties are attached to a providedcompound via click chemistry. In some embodiments, such moieties areattached via a 1,3-cycloaddition of an azide with an alkyne, optionallyin the presence of a copper catalyst. Methods of using click chemistryare known in the art and include those described by Rostovtsev et al.,Angew. Chem. Int. Ed. 2002, 41, 2596-99 and Sun et al., BioconjugateChem., 2006, 17, 52-57. In some embodiments, a click ready inhibitormoiety is provided and reacted with a click ready -T-R¹ moiety. As usedherein, “click ready” refers to a moiety containing an azide or alkynefor use in a click chemistry reaction. In some embodiments, the clickready inhibitor moiety comprises an azide. In certain embodiments, theclick ready -T-R¹ moiety comprises a strained cyclooctyne for use in acopper-free click chemistry reaction (for example, using methodsdescribed in Baskin et al., Proc. Natl. Acad. Sci. USA 2007, 104,16793-16797).

In some embodiments, the detectable moiety, R¹, is selected from alabel, a dye, a photocrosslinker, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, an antibodyor antibody fragment, a biomaterial, a nanoparticle, a spin label, afluorophore, a metal-containing moiety, a radioactive moiety, quantumdot(s), a novel functional group, a group that covalently ornoncovalently interacts with other molecules, a photocaged moiety, anactinic radiation excitable moiety, a ligand, a photoisomerizablemoiety, biotin, a biotin analog (e.g., biotin sulfoxide), a moietyincorporating a heavy atom, a chemically cleavable group, aphotocleavable group, a redox-active agent, an isotopically labeledmoiety, a biophysical probe, a phosphorescent group, a chemiluminescentgroup, an electron dense group, a magnetic group, an intercalatinggroup, a chromophore, an energy transfer agent, a biologically activeagent, a detectable label, or a combination thereof.

In some embodiments, R^(t) is biotin or an analog thereof. In certainembodiments, R^(t) is biotin. In certain other embodiments, R^(t) isbiotin sulfoxide.

In another embodiment, R^(t) is a fluorophore. In a further embodiment,the fluorophore is selected from Alexa Fluor dyes (Alexa Fluor 350,Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568,Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680),AMCA, AMCA-S, BODIPY dyes (BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR,BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665),Carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue, CascadeYellow, Coumarin 343, Cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5), Dansyl,Dapoxyl, Dialkylaminocoumarin,4′,5′-Dichloro-2′,7′-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin,Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800),JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin,Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, RhodamineGreen, Rhodamine Red, Rhodol Green,2′,4′,5′,7′-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR),Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X,5(6)-Carboxyfluorescein, 2,7-Dichlorofluorescein,N,N-Bis(2,4,6-trimethylphenyl)-3,4:9,10-perylenebis(dicarboximide, HPTS,Ethyl Eosin, DY-490XL MegaStokes, DY-485XL MegaStokes, Adirondack Green520, ATTO 465, ATTO 488, ATTO 495, YOYO-1,5-FAM, BCECF,dichlorofluorescein, rhodamine 110, rhodamine 123, YO—PRO-1, SYTOXGreen, Sodium Green, SYBR Green I, Alexa Fluor 500, FITC, Fluo-3,Fluo-4, fluoro-emerald, YoYo-1 ssDNA, YoYo-1 dsDNA, YoYo-1, SYTORNASelect, Diversa Green-FP, Dragon Green, EvaGreen, Surf Green EX,Spectrum Green, NeuroTrace 500525, NBD-X, MitoTracker Green FM,LysoTracker Green DND-26, CBQCA, PA-GFP (post-activation), WEGFP(post-activation), F1ASH-CCXXCC, Azami Green monomeric, Azami Green,green fluorescent protein (GFP), EGFP (Campbell Tsien 2003), EGFP(Patterson 2001), Kaede Green,7-Benzylamino-4-Nitrobenz-2-Oxa-1,3-Diazole, Bexl, Doxorubicin, LumioGreen, or SuperGlo GFP.

As described generally above, a provided probe compound comprises atethering moiety, -T-, that attaches the irreversible inhibitor to thedetectable moiety. As used herein, the term “tether” or “tetheringmoiety” refers to any bivalent chemical spacer. Exemplary tethers are acovalent bond, a polymer, a water soluble polymer, optionallysubstituted alkyl, optionally substituted heteroalkyl, optionallysubstituted heterocycloalkyl, optionally substituted cycloalkyl,optionally substituted heterocyclyl, optionally substitutedheterocycloalkylalkyl, optionally substituted heterocycloalkylalkenyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocycloalkylalkenylalkyl, an optionallysubstituted amide moiety, an ether moiety, an ketone moiety, an estermoiety, an optionally substituted carbamate moiety, an optionallysubstituted hydrazone moiety, an optionally substituted hydrazinemoiety, an optionally substituted oxime moiety, a disulfide moiety, anoptionally substituted imine moiety, an optionally substitutedsulfonamide moiety, a sulfone moiety, a sulfoxide moiety, a thioethermoiety, or any combination thereof.

In some embodiments, the tethering moiety, -T¹-, is selected from acovalent bond, a polymer, a water soluble polymer, optionallysubstituted alkyl, optionally substituted heteroalkyl, optionallysubstituted heterocycloalkyl, optionally substituted cycloalkyl,optionally substituted heterocycloalkylalkyl, optionally substitutedheterocycloalkylalkenyl, optionally substituted aryl, optionallysubstituted heteroaryl, and optionally substitutedheterocycloalkylalkenylalkyl. In some embodiments, the tethering moietyis an optionally substituted heterocycle. In other embodiments, theheterocycle is selected from aziridine, oxirane, episulfide, azetidine,oxetane, pyrroline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine,pyrazole, pyrrole, imidazole, triazole, tetrazole, oxazole, isoxazole,oxirene, thiazole, isothiazole, dithiolane, furan, thiophene,piperidine, tetrahydropyran, thiane, pyridine, pyran, thiapyrane,pyridazine, pyrimidine, pyrazine, piperazine, oxazine, thiazine,dithiane, and dioxane. In some embodiments, the heterocycle ispiperazine. In further embodiments, the tethering moiety is optionallysubstituted with halogen, —CN, —OH, —NO₂, alkyl, S(O), and S(O)₂. Inother embodiments, the water soluble polymer is a PEG group.

In other embodiments, the tethering moiety provides sufficient spatialseparation between the detectable moiety and the protein kinaseinhibitor moiety. In further embodiments, the tethering moiety isstable. In yet a further embodiment, the tethering moiety does notsubstantially affect the response of the detectable moiety. In otherembodiments, the tethering moiety provides chemical stability to theprobe compound. In further embodiments, the tethering moiety providessufficient solubility to the probe compound.

In some embodiments, a tethering moiety, -T¹-, such as a water solublepolymer is coupled at one end to a provided irreversible inhibitor andto a detectable moiety, R^(t), at the other end. In other embodiments, awater soluble polymer is coupled via a functional group or substituentof the provided irreversible inhibitor. In further embodiments, a watersoluble polymer is coupled via a functional group or substituent of thereporter moiety.

In some embodiments, examples of hydrophilic polymers, for use intethering moiety -T¹-, include, but are not limited to: polyalkyl ethersand alkoxy-capped analogs thereof (e.g., polyoxyethylene glycol,polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogsthereof, polyoxyethylene glycol, the latter is also known aspolyethylene glycol or PEG); polyvinylpyrrolidones; polyvinylalkylethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyloxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkylacrylamides (e.g., polyhydroxypropylmethacrylamide and derivativesthereof); polyhydroxyalkyl acrylates; polysialic acids and analogsthereof, hydrophilic peptide sequences; polysaccharides and theirderivatives, including dextran and dextran derivatives, e.g.,carboxymethyldextran, dextran sulfates, aminodextran; cellulose and itsderivatives, e.g., carboxymethyl cellulose, hydroxyalkyl celluloses;chitin and its derivatives, e.g., chitosan, succinyl chitosan,carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and itsderivatives; starches; alginates; chondroitin sulfate; albumin; pullulanand carboxymethyl pullulan; polyaminoacids and derivatives thereof,e.g., polyglutamic acids, polylysines, polyaspartic acids,polyaspartamides; maleic anhydride copolymers such as: styrene maleicanhydride copolymer, divinylethyl ether maleic anhydride copolymer;polyvinyl alcohols; copolymers thereof, terpolymers thereof, mixturesthereof, and derivatives of the foregoing. In other embodiments, a watersoluble polymer is any structural form. Exemplary forms are linear,forked or branched. In further embodiments, multifunctional polymerderivatives include, but are not limited to, linear polymers having twotermini, each terminus being bonded to a functional group which is thesame or different.

In some embodiments, a water polymer comprises a poly(ethylene glycol)moiety. In further embodiments, the molecular weight of the polymer isof a wide range. Exemplary ranges are between about 100 Da and about100,000 Da or more. In yet further embodiments, the molecular weight ofthe polymer is between about 100 Da and about 100,000 Da, about 100,000Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da,about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da,about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da,about 35,000 Da, 30,000 Da, about 25,000 Da, about 20,000 Da, about15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da,about 2,000 Da, about 1,000 Da, about 900 Da, about 800 Da, about 700Da, about 600 Da, about 500 Da, about 400 Da, about 300 Da, about 200Da, and about 100 Da. In some embodiments, the molecular weight of thepolymer is between about 100 Da and 50,000 Da. In some embodiments, themolecular weight of the polymer is between about 100 Da and 40,000 Da.In some embodiments, the molecular weight of the polymer is betweenabout 1,000 Da and 40,000 Da. In some embodiments, the molecular weightof the polymer is between about 5,000 Da and 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 10,000Da and 40,000 Da. In some embodiments, the polyethylene glycol) moleculeis a branched polymer. In further embodiments, the molecular weight ofthe branched chain PEG is between about 1,000 Da and about 100,000 Da.Exemplary ranges are about 100,000 Da, about 95,000 Da, about 90,000 Da,about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da,about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da,about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da,about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da,about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, and about1,000 Da. In some embodiments, the molecular weight of a branched chainPEG is between about 1,000 Da and about 50,000 Da. In some embodiments,the molecular weight of a branched chain PEG is between about 1,000 Daand about 40,000 Da. In some embodiments, the molecular weight of abranched chain PEG is between about 5,000 Da and about 40,000 Da. Insome embodiments, the molecular weight of a branched chain PEG isbetween about 5,000 Da and about 20,000 Da. The foregoing list forsubstantially water soluble backbones is by no means exhaustive and ismerely illustrative, and in some embodiments, polymeric materials havingthe qualities described above are suitable for use in methods andcompositions described herein.

One of ordinary skill in the art will appreciate that when -T¹-R^(t) isattached to a compound of the formulae herein via the R² group, then theresulting tethering moiety comprises the R² group.

In certain embodiments, the tethering moiety, -T¹-, has one of thefollowing structures:

In some embodiments, the tethering moiety, -T¹-, has the followingstructure:

In some embodiments, the tethering moiety, -T¹-, has the followingstructure:

In other embodiments, the tethering moiety, -T¹-, has the followingstructure:

In certain other embodiments, the tethering moiety, -T¹-, has thefollowing structure:

In yet other embodiments, the tethering moiety, -T¹-, has the followingstructure:

In some embodiments, the tethering moiety, -T¹-, has the followingstructure:

In some embodiments, -T¹-R^(t) is of the following structure:

In some embodiments, -T¹-R^(t) is of the following structure:

In other embodiments, -T¹-R^(t) is of the following structure:

In certain embodiments, -T¹-R^(t) is of the following structure:

In some embodiments, a probe compound of formula I-t is derived from anycompound described herein.

In certain embodiments, the probe compound is one of the followingstructures:

It will be appreciated that many -T¹-R^(t) reagents are commerciallyavailable. For example, numerous biotinylating reagents are availablefrom, e.g., Thermo Scientific having varying tether lengths. Suchreagents include NHS-PEG₄-Biotin and NHS-PEG₁₂-Biotin,

In some embodiments, analogous probe structures to the ones exemplifiedabove are prepared using click-ready inhibitor moieties and click-ready-T¹-R^(t) moieties, as described herein.

In some embodiments, a provided probe compound covalently modifies aphosphorylated conformation of a protein kinase. In one aspect, thephosphorylated conformation of the protein kinase is either an active orinactive form of the protein kinase. In certain embodiments, thephosphorylated conformation of the protein kinase is an active form ofsaid kinase. In certain embodiments, the probe compound is cellpermeable.

In some embodiments, the present invention provides a method fordetermining occupancy of a protein kinase by a provided irreversibleinhibitor (i.e., a compound of any of the formulae presented herein) ina patient, comprising providing one or more tissues, cell types, or alysate thereof, obtained from a patient administered at least one doseof a compound of said irreversible inhibitor, contacting said tissue,cell type or lysate thereof with a probe compound (i.e., a compound offormula I-t) to covalent modify at least one protein kinase present insaid lysate, and measuring the amount of said protein kinase covalentlymodified by the probe compound to determine occupancy of said proteinkinase by said compound as compared to occupancy of said protein kinaseby said probe compound. In certain embodiments, the method furthercomprises the step of adjusting the dose of the compound of formulaepresented herein to increase occupancy of the protein kinase. In certainother embodiments, the method further comprises the step of adjustingthe dose of the compound of formulae presented herein to decreaseoccupancy of the protein kinase.

As used herein, the terms “occupancy” or “occupy” refer to the extent towhich a protein kinase is modified by a provided covalent inhibitorcompound. One of ordinary skill in the art would appreciate that it isdesirable to administer the lowest dose possible to achieve the desiredefficacious occupancy of the protein kinase.

In some embodiments, the protein kinase to be modified is FGFR4.

In some embodiments, the probe compound comprises the irreversibleinhibitor for which occupancy is being determined.

In some embodiments, the present invention provides a method forassessing the efficacy of a provided irreversible inhibitor in a mammal,comprising administering a provided irreversible inhibitor to themammal, administering a provided probe compound to tissues or cellsisolated from the mammal, or a lysate thereof, measuring the activity ofthe detectable moiety of the probe compound, and comparing the activityof the detectable moiety to a standard.

In other embodiments, the present invention provides a method forassessing the pharmacodynamics of a provided irreversible inhibitor in amammal, comprising administering a provided irreversible inhibitor tothe mammal, administering a probe compound presented herein to one ormore cell types, or a lysate thereof, isolated from the mammal, andmeasuring the activity of the detectable moiety of the probe compound atdifferent time points following the administration of the inhibitor.

In yet other embodiments, the present invention provides a method for invitro labeling of a protein kinase comprising contacting said proteinkinase with a probe compound described herein. In one embodiment, thecontacting step comprises incubating the protein kinase with a probecompound presented herein.

In certain embodiments, the present invention provides a method for invitro labeling of a protein kinase comprising contacting one or morecells or tissues, or a lysate thereof, expressing the protein kinasewith a probe compound described herein.

In certain other embodiments, the present invention provides a methodfor detecting a labeled protein kinase comprising separating proteins,the proteins comprising a protein kinase labeled by probe compounddescribed herein, by electrophoresis and detecting the probe compound byfluorescence.

In some embodiments, the present invention provides a method forassessing the pharmacodynamics of a provided irreversible inhibitor invitro, comprising incubating the provided irreversible inhibitor withthe target protein kinase, adding the probe compound presented herein tothe target protein kinase, and determining the amount of target modifiedby the probe compound.

In certain embodiments, the probe compound is detected by binding toavidin, streptavidin, neutravidin, or captavidin.

In some embodiments, the probe is detected by Western blot. In otherembodiments, the probe is detected by ELISA. In certain embodiments, theprobe is detected by flow cytometry.

In other embodiments, the present invention provides a method forprobing the kinome with irreversible inhibitors comprising incubatingone or more cell types, or a lysate thereof, with a biotinylated probecompound to generate proteins modified with a biotin moiety, digestingthe proteins, capturing with avidin or an analog thereof, and performingmulti-dimensional LC-MS-MS to identify protein kinases modified by theprobe compound and the adduction sites of said kinases.

In certain embodiments, the present invention provides a method formeasuring protein synthesis in cells comprising incubating cells with anirreversible inhibitor of the target protein, forming lysates of thecells at specific time points, and incubating said cell lysates with aninventive probe compound to measure the appearance of free protein overan extended period of time.

In other embodiments, the present invention provides a method fordetermining a dosing schedule in a mammal for maximizing occupancy of atarget protein kinase comprising assaying a one or more cell types, or alysate thereof, isolated from the mammal, (derived from, e.g.,splenocytes, peripheral B cells, whole blood, lymph nodes, intestinaltissue, or other tissues) from a mammal administered a providedirreversible inhibitor of any of the formulae presented herein, whereinthe assaying step comprises contacting said one or more tissues, celltypes, or a lysate thereof, with a provided probe compound and measuringthe amount of protein kinase covalently modified by the probe compound.

Exemplification

As depicted in the Examples below, in certain exemplary embodiments,compounds are prepared according to the following general procedures. Itwill be appreciated that, although the general methods depict thesynthesis of certain compounds of the present invention, the followinggeneral methods, and other methods known to one of ordinary skill in theart, can be applied to all compounds and subclasses and species of eachof these compounds, as described herein.

Compound numbers utilized in the Examples below correspond to compoundnumbers set forth supra.

Example 1: Synthesis of Common Intermediate 6

Step 1: Intermediate 1

A 1-L, three-necked flask equipped with a mechanical stirrer, wascharged with uracil (45.0 g, 401 mmol) and paraformaldehyde (14.5 g, 483mmol). A solution of potassium hydroxide (0.5 M, 600 mL, 0.30 mol) wasadded in one portion. The resulting mixture was stirred at 55° C.overnight. The mixture was cooled in an ice-water bath and the pH wasadjusted to 6 with 12 N HCl. The resulting precipitate was collected byfiltration and dried to afford the title compound (46.0 g) as a whitesolid which was used in the next step without further purification. ¹HNMR (400 MHz, DMSO-d6): δ 4.12 (d, 2H), 4.78 (t, 1H), 7.24 (s, 1H),10.64 (s, 1H), 10.98 (s, 1H).

Step 2: Intermediate 2

A 500-mL, three-necked flask equipped with a mechanical stirrer wascharged with Intermediate 1 (25.0 g, 176 mmol), toluene (30 mL), andphosphorous oxychloride (125 mL). DIPEA (130 mL) was added dropwise over10 min. The resulting mixture was heated at reflux overnight. Thesolution was concentrated and the resulting residue was slowly pouredonto cooled (0° C.) 1.5 M HCl and extracted with EtOAc. The organicphase was washed with water, saturated aqueous NaHCO₃, brine, dried overanhydrous Na₂SO₄ and concentrated to afford the title compound (32.0 g)which was used in the next step without further purification. ¹H NMR(400 MHz, CDCl₃): δ 4.64 (s, 2H), 8.66 (s, 1H).

Step 3: Intermediate 3

500-mL, three-necked flask equipped with a mechanical stirrer wascharged with Intermediate 2 (32.0 g, 111 mmol), acetone (150 mL), NaI(26.5 g, 177 mmol). The mixture was allowed to stir at ambienttemperature for 15 min then was heated at reflux for 30 min. The mixturewas cooled to ambient temperature and filtered to remove the resultantsolid. The filtrate was concentrated to afford 46.0 g of the titlecompound which was used in the next step without further purification.¹H NMR (400 MHz, CDCl₃): δ 4.39 (s, 2H), 8.60 (s, 1H).

Step 4: Intermediate 4

A mixture of Intermediate 3 (15.0 g, 47.9 mmol), 3,5-dimethoxyaniline(8.80 g, 57.4 mmol), and K₂CO₃ (14.4 g, 104 mmol) in acetone (150 mL)was stirred at ambient temperature overnight. The solution was cooled inan ice-water bath and filtered to remove the resultant solid. Thefiltrate was concentrated and the residue was triturated with EtOH (100mL) then stirred at 0° C. for 30 min. The precipitate was collected byfiltration and dried to afford 9.40 g of the title compound. MS m/z:314.2 (M+H⁺). ¹H NMR (400 MHz, CDCl₃): δ 3.73 (s, 6H), 4.22 (br s, 1H),4.40 (s, 2H), 5.74 (d, 2H), 5.94 (t, 1H), 8.53 (s, 1H).

Step 5: Intermediate 5

A solution of Intermediate 4 (9.40 g, 29.9 mmol), DIPEA (9.60 g, 74.3mmol), and MeNH₂HCl (2.40 g, 35.8 mmol) in dioxane (150 mL) was stirredin sealed tube at 60° C. overnight. The solution was cooled to ambienttemperature. DIPEA (9.60 g, 74.3 mmol) was added followed by slowaddition of triphosgene (9.30 g, 31.3 mmol) in dioxane (60 mL). Thesolution was allowed to stir at ambient temperature for 1 h after whichit was heated to 70° C. for 3 h. The solution was concentrated, waterwas added, and the mixture was allowed to stir for 30 min at ambienttemperature. The resultant solid was collected by filtration then wasdissolved in MeOH/H₂O (135 mL/15 mL) and heated at reflux for 10 minafter which, the solution was cooled in ice-water bath and the resultantsolid was collected by filtration, washed with cold MeOH/H₂O (v/v: 18/2)and dried to afford the title compound (5.80 g) which was used in thenext step without further purification. MS m/z: 335.3 (M+H⁺). ¹H NMR(400 MHz, CDCl₃): δ 3.46 (s, 3H), 3.79 (s, 6H), 4.74 (s, 2H), 6.41 (t,1H), 6.46 (d, 2H), 8.12 (s, 1H).

Step 6: Common Intermediate 6

A solution of Intermediate 5 (5.50 g, 16.4 mmol) in DCM (150 mL) wascooled to 0° C. and SO₂Cl₂ (4.70 g, 34.8 mmol) in DCM (20 mL) was addeddropwise. The resulting mixture was allowed to stir at 0° C. for 1 hafter which it was poured into saturated aqueous NaHCO₃ and the organicphase was separated, washed with water, brine, dried over Na₂SO₄ andconcentrated to afford 6.00 g of the title compound. MS m/z: 403.3(M+H⁺). ¹H NMR (400 MHz, CDCl₃): δ 3.47 (s, 3H), 3.95 (s, 6H), 4.65 (s,2H), 6.62 (s, 1H), 8.12 (s, 1H).

Example 2: Synthesis of Common Intermediate 8

Step 1: Intermediate 2

Intermediate 2 was prepared according to literature procedures fromIntermediate 1 (EP2112150 A1, 2009).

Step 2: Intermediate 3

To a suspension of Intermediate 2 (2.10 g, 11.0 mmol) in 140 mL of1,4-dioxane was added 3-nitrobenzylamine hydrochloride (2.49 g, 13.2mmol) and triethylamine (5.22 mL, 37.5 mmol). The mixture was allowed tostir at 95° C. for 24 h. Additional 3-nitrobenzylamine hydrochloride(208 mg, 1.10 mmol) and triethylamine (3.08 mL, 22.1 mmol) were addedand the reaction was stirred at 100° C. for 17 h. The reaction mixturewas concentrated and the crude product was subjected to flashchromatography on silica gel (eluting with a gradient of 50-100% EtOAcin heptane). The resulting residue was then triturated with EtOAc toprovide 2.05 g of the title compound. MS m/z: 307.0 (M+H)⁺. ¹H NMR (400MHz, DMSO-d6): δ: 8.18 (1H, s), 7.85 (1H, s), 7.74 (1H, d), 7.59 (2H,m), 5.11 (1H, t), 4.66 (2H, d), 4.35 (2H, d), 2.28 (3H, s).

Step 3: Intermediate 4

To a solution of Intermediate 3 (2.10 g, 6.69 mmol) in 140 mL of DCM wasadded manganese oxide (8.02 g, 53.5 mmol) and the mixture was stirred atambient temperature for 17 h. The reaction mixture was filtered throughCelite, washed with DCM and the filtrate concentrated under reducedpressure to give 1.85 g of the title compound, which was used directlywithout purification. MS m/z: 305.1 (M+H⁺). ¹H NMR (400 MHz, CDCl₃): δ:9.75 (1H, s), 9.08 (1H, br s), 8.38 (1H, s), 8.20 (1H, s), 8.15 (1H, d),7.66 (1H, d), 7.52 (1H, t), 4.87 (2H, d), 2.48 (3H, s).

Step 4: Intermediate 5

To a solution of Intermediate 4 (500 mg, 1.64 mmol) in 9 mL of DCM wasadded 3,5-dimethoxyaniline (229 mg, 1.49 mmol) and acetic acid (94.1 μL,1.64 mmol) under argon. The mixture was allowed to stir at ambienttemperature for 15 min prior to the addition of sodiumtriacetoxyborohydride (2×238 mg, 2.24 mmol) in 2 portions with a 15 mininterval in between. The reaction was stirred at ambient temperature for17 h, extra sodium triacetoxyborohydride (476 mg, 2.24 mmol) was addedand the mixture was stirred for an additional 6 h. The reaction wasquenched with 10 mL of 1 M NaOH which caused vigorous evolution of gasafter which the mixture was stirred for 15 min. The aqueous layer wasextracted with DCM (×3) and the combined organic layers were washed withbrine, dried (Na₂SO₄), filtered and concentrated. The crude product wassubjected to flash chromatography on silica gel (eluting with 50% EtOAcin heptane), which gave 591 mg of title compound. MS m/z: 442.1 (M+H)⁺.

Step 5: Intermediate 6

To a solution of Intermediate 5 (591 mg, 1.34 mmol) in 6 mL of 2-MeTHFwas added triphosgene (437 mg, 1.47 mmol) followed by slow addition oftriethylamine (578 μL, 4.15 mmol) under argon. The mixture was stirredat ambient temperature for 1 h, then at 70° C. for 1.5 h. The reactionwas quenched with a 1:1 mixture of saturated aqueous NaHCO₃/H₂O (12 mL)and the resultant solids were removed by filtration and washed withEtOAc. The filtrate layers were separated and the aqueous was extractedwith EtOAc followed by DCM. The combined organic layers were washed withbrine, dried (Na₂SO₄), filtered and concentrated in vacuo. The crudeproduct was subjected to dry-flash chromatography on silica gel (elutingwith a gradient of 50-70% EtOAc in heptane). The resultant residue wasthen triturated with Et₂O to provide 398 mg of the title compound. MSm/z: 468.0 (ES+, M+H⁺). ¹H NMR (400 MHz, CDCl₃): δ: 8.34 (1H, s), 8.11(2H, m), 7.80 (1H, d), 7.47 (1H, t), 6.47 (2H, d), 6.40 (1H, t), 5.38(2H, s), 4.73 (2H, s), 3.77 (6H, s), 2.52 (3H, s).

Step 6: Intermediate 7

To a cooled (0° C.) solution of Intermediate 6 (396 mg, 0.847 mmol) in 7mL of MeCN and 15 mL of DCM was added sulfuryl chloride (137 μL, 1.69mmol). The reaction was stirred at 0° C. for 15 min. The reaction wasquenched with saturated aqueous NaHCO₃ and the aqueous was extractedwith DCM (×2). The combined organic layers were dried (Na₂SO₄), filteredand concentrated in vacuo to give 452 mg of the title product which wasused directly without purification. MS m/z: 536.0 (H⁺). ¹H NMR (400 MHz,CDCl₃): δ: 8.32 (1H, s), 8.09 (2H, m), 7.78 (1H, d), 7.46 (1H, t), 6.61(1H, s), 5.38 (2H, s), 4.65 (2H, s), 3.94 (6H, s), 2.49 (3H, s).

Step 7: Intermediate 8

To a solution of Intermediate 7 (451 mg, 0.841 mmol) in 10 mL of DCM wasadded mCPBA (228 mg, 0.925 mmol). The reaction was stirred at ambienttemperature for 1 hour and then quenched with 6 mL of saturated aqueousNaHCO₃ and 4 mL of 2 M sodium thiosulfate. The mixture was stirred for15 min, then diluted with H₂O and the aqueous was extracted with DCM(×3). The combined organic layers were dried (Na₂SO₄), filtered andconcentrated to give 468 mg of the title product which was used directlywithout purification. MS m/z: 552.0 M+H⁺).

Example 3: Synthesis of I-1

Step 1: Intermediate 2

To a solution of Intermediate 8 from Example 2 (150 mg, 0.27 mmol), in1,4-dioxane (8 mL) was added benzene-1,2-diamine (88.1 mg, 0.82 mmol)and p-toluene sulfonic acid (23.4 mg, 0.14 mmol). The mixture was heatedat 100° C. overnight under nitrogen. The mixture was cooled to ambienttemperature, water was added and the resultant suspension was filteredto afford 145 mg of the title compound. MS m/z: 596.3 (M+H)⁺.

Step 2: Intermediate 3

To a solution of Intermediate 2 (145 mg, 0.24 mmol) in THF (15 mL) wasadded triethylamine (98.0 mg, 0.97 mmol) and (Boc)₂O (106 mg, 0.48mmol). The reaction mixture was heated at reflux overnight. The mixturewas cooled to ambient temperature, water was added, and the aqueouslayer extracted with EtOAc (30 mL×3). The combined organic layers weredried (Na₂SO₄), filtered, and concentrated. The crude product wassubjected to flash chromatography on silica gel (65% EtOAc/hexanes) toafford 121 mg of the title compound. ¹H NMR (400 MHz, DMSO-d6): δ 1.44(s, 9H), 3.97 (s, 6H), 4.62 (s, 2H), 5.16 (s, 2H), 6.96-7.11 (m, 3H),7.39 (d, 1H), 7.47-7.57 (m, 3H), 8.04-8.07 (m, 2H), 8.16 (s, 1H), 8.48(s, 1H), 8.58 (s, 1H).

Step 3: Intermediate 4

To a solution of Intermediate 3 (112 mg, 0.16 mmol) in ethanol (8 mL)and water (4 mL) was added iron power (54.0 mg, 0.96 mmol) and NH₄Cl (52mg, 0.96 mmol). The mixture was heated at reflux for 1.5 h. The reactionmixture was cooled to ambient temperature, filtered, and the filtratewas concentrated. Water was added to the resultant residue and theaqueous layer was and extracted with EtOAc (30 mL×3). The combinedorganic layers were washed with saturated NaHCO₃, dried (Na₂SO₄),filtered and concentrated to afford 104.4 mg of the title compound whichwas used directly in the next step without purification. MS m/z: 666.4[M+H]⁺

Step 4: Intermediate 5

A mixture of Intermediate 4 (98.6 mg, 0.15 mmol), propionic acid (16.4mg, 0.22 mmol) and HATU (113 mg, 0.30 mmol) in DMF (6 mL) was cooled to0° C. DIPEA (57.3 mg, 0.44 mmol) was added and the reaction mixture wasallowed to stir at ambient temperature overnight. Water was added andthe resultant mixture was extracted with EtOAc (60 mL×3). The combinedorganic layers were dried (Na₂SO₄), filtered, and concentrated. Thecrude product was subjected to flash chromatography on silica gel (3%MeOH/DCM) to afford 74 mg of the title compound. MS m/z: 722.4 [M+H]⁺

Step 5: Intermediate 6

To a solution of Intermediate 5 (74.0 mg, 0.10 mmol) in DCM (2 mL) wasadded TFA (2 mL) and the solution was allowed to stir at ambienttemperature for 30 min. The solvent was removed and water was added tothe residue which was extracted with EtOAc (30 mL×3). The combinedorganic layers were washed with saturated NaHCO₃, dried (Na₂SO₄),filtered and concentrated to afford 79 mg of the title compound whichwas used without purification. MS m/z: 622.4 [M+H]⁺

Step 6: I-1

To a cooled (0° C.) solution of Intermediate 6 (79.0 mg, 0.13 mmol) andDIPEA (33.0 mg, 0.25 mmol) in THF (3 mL) was added acryloyl chloride(13.8 mg, 0.15 mmol). The reaction was stirred at 0° C. for 10 min. Thereaction was quenched with saturated aqueous NaHCO₃ and extracted withEtOAc (30 mL×3). The combined organic layers were washed with saturatedaqueous NaHCO₃, dried (Na₂SO₄), filtered and concentrated. The crudeproduct was subjected to flash chromatography on silica gel (5%MeOH/DCM) to 34.3 mg of the title compound. MS m/z: 676.4 [M+H]⁺. ¹H NMR(400 MHz, DMSO-d6): δ 1.08 (t, 3H), 2.29 (q, 2H), 3.96 (s, 6H), 4.60 (s,2H), 5.06 (s, 2H), 5.68-5.83 (m, 1H), 6.26 (dd, 1H), 6.40-6.56 (m, 1H),6.83 (d, 1H), 6.95-7.09 (m, 3H), 7.16 (t, 1H), 7.37-7.48 (m, 1H),7.50-7.53 (m, 3H), 8.14 (s, 1H), 8.53 (s, 1H), 9.70 (s, 1H), 9.74 (s,1H).

Example 4: Synthesis of I-2

Step 1: Intermediate 2

A mixture of Intermediate 1 (prepared as described in Example 2 usingmethylamine in place of 3-nitrobenzylamine in Step 2 (106 mg, 0.25mmol)), benzene-1,2-diamine (80 mg, 0.74 mmol), and p-TsOH (21.2 mg,0.12 mmol) in 1,4-dioxane (8 mL) was heated at reflux for 16 h undernitrogen. The reaction mixture was cooled to ambient temperature andpartitioned between EtOAc and water. The organic phase was washed withsaturated aqueous NaHCO₃, brine, dried (Na₂SO₄) and concentrated invacuo. The crude product was purified with column chromatography onsilica gel (5% MeOH/DCM) to afford the title compound (69.3 mg). MS m/z:475.3 (M+H)⁺. ¹H NMR (400 MHz, DMSO-d6): δ 3.23 (s, 3H), 3.97 (s, 6H),4.50 (s, 2H), 4.81 (s, 2H), 6.57 (t, 1H), 6.70-6.79 (m, 1H), 6.83-6.94(m, 1H), 6.99 (s, 1H), 7.35 (d, 1H), 8.04 (s, 1H), 8.45 (s, 1H).

Step 2: I-2

To a cooled (0° C.) solution of Intermediate 2 (69.1 mg, 0.15 mmol) andDIPEA (37.4 mg, 0.29 mmol) in THF (10 mL) was added acryloyl chloride(15.8 mg, 0.17 mmol). The reaction was allowed to stir at 0° C. for 10min. The reaction was quenched with saturated aqueous NaHCO₃ andextracted with EtOAc (40 mL×3). The combined organic layers were washedwith saturated aqueous NaHCO₃, dried (Na₂SO₄), filtered and concentratedin vacuo. The crude product was purified by column chromatography onsilica gel (3% MeOH/DCM) to afford I-2 (23.3 mg). MS m/z: 529.3 (M+H)⁺.¹H NMR (400 MHz, DMSO-d6): δ 3.22 (s, 3H), 3.96 (s, 6H), 4.52 (s, 2H),5.77 (dd, 2.0 Hz, 1H), 6.27 (dd, 2.0 Hz, 1H), 6.47-6.54 (m, 1H), 6.99(s, 1H), 7.108-7.13 (m, 1H), 7.17-7.22 (m, 1H), 7.56 (d, 1H), 7.80 (d,1H), 8.09 (s, 1H), 8.59 (s, 1H), 9.76 (s, 1H).

Example 5: Synthesis of I-3

Step 1: Intermediate 2

To a mixture of Intermediate 1 (prepared as described in Example 1 usingbenzylamine in place of methylamine in Step 4 (54.0 mg, 0.11 mmol)), wasadded benzene-1,2-diamine (24.0 mg, 0.22 mmol) in 1,4-dioxane (2 mL) andone drop of TFA. The reaction mixture was heated at reflux in a sealedtube for 16 h after which it was cooled to ambient temperature andpartitioned between EtOAc and water. The organic phase was washed withsaturated aqueous NaHCO₃ brine, dried (Na₂SO₄) and concentrated invacuo. The crude product was purified with column chromatography onsilica gel (eluting with 100% EtOAc) to afford the title compound (34.5mg). MS m/z: 551.0 (M+H)⁺.

Step 2: I-3

To a cooled (0° C.) solution of Intermediate 2 (14.0 mg, 0.025 mmol) inTHF (1 mL) was added acryloyl chloride (1.9 uL, 0.023 mmol). Thereaction was stirred at 0° C. for 10 min. after which it was purified byreverse phase HPLC (eluting with a gradient of 0-90% MeCN in water).Combined fractions were stirred with saturated aqueous NaHCO₃, extractedwith DCM, and the organic layers concentrated to afford 10.6 mg of thetitle compound. MS m/z: 605.2 (M+H)⁺.

Example 6: Synthesis of I-4

The title compound was prepared as outlined in Example 3 usingtert-butyl 3-aminobenzylcarbamate in place of benzene-1,2-diamine inStep 1. MS m/z: 690.2 (M+H⁺). ¹H NMR (400 MHz, CD₃OD): δ: 8.07 (1H, s),7.63 (1H, s), 7.45 (1H, s), 7.32 (2H, m), 7.23 (2H, m), 7.00 (2H, m),6.90 (1H, s), 6.26 (2H, m), 5.66 (1H, m), 5.28 (2H, s), 4.68 (2H, s),4.33 (2H, s), 3.96 (6H, s), 2.34 (2H, q), 1.89 (3H, t).

Example 7: Synthesis of I-5

Step 1: Intermediate 2

To a mixture of Intermediate 5 (30.1 mg, 0.09 mmol) from Example 1 andbenzene-1,2-diamine (19.5 mg, 0.18 mmol) in 1,4-dioxane (1 mL) was addedone drop of TFA. The reaction mixture was heated at reflux in a sealedtube for 16 h after which it was cooled to ambient temperature andpartitioned between EtOAc and water. The organic phase was washed withsaturated aqueous NaHCO₃ brine, dried (Na₂SO₄) and concentrated invacuo. The crude product was purified through column chromatography onsilica gel (eluting with 100% EtOAc) to afford the title compound (16.7mg). MS m/z: 551.0 (M+H)⁺.

Step 2: I-5

To a cooled (0° C.) solution of Intermediate 2 (14.0 mg, 0.034 mmol) inTHF (1 mL) was added acryloyl chloride (2.8 uL, 0.034 mmol). Thereaction was stirred at 0° C. for 10 min. after which it was purified byreverse phase HPLC (eluting with a gradient of 0-90% MeCN in water with0.1% TFA). Combined fractions were concentrated, stirred with saturatedaqueous NaHCO₃, extracted with DCM, and the organic layers concentratedto afford 14.6 mg of the title compound. MS m/z: 407.2 (M+H)⁺. ¹H NMR(400 MHz, DMSO-d6): δ 3.23 (s, 3H), 3.75 (s, 6H), 4.72 (s, 2H), 5.79(dd, 1H), 6.28 (dd, 1H), 6.56-6.45 (m, 2H), 6.60 (s, 2H), 7.25-7.27 (m,2H), 7.59 (d, 1H), 7.72 (d, 1H), 8.09 (s, 1H), 9.41 (br s, 1H), 9.88 (s,1H).

Example 8: Synthesis of I-6

Compound I-6 was prepared as described in Example 5 using tert-butyl(2-amino-4-(trifluoromethyl)phenyl)carbamate instead ofbenzene-1,2-diamine in Step 1. MS m/z: 673.1 (M+H)⁺

Example 9: Synthesis of I-7, (Racemic)

Step 1: Intermediate 2

To a solution of Intermediate 8 from Example 2 (150 mg, 0.27 mmol) indioxane (15 mL) was added trans-cyclohexane-1,2-diamine (62 mg, 0.54mmol) and catalytic p-toluene sulfonic acid. The resultant mixture washeated at reflux for 16 h. The reaction mixture was partitioned betweenEtOAc and saturated aqueous NaHCO₃. The organic layer was separated,dried (Na₂SO₄) and evaporated to dryness. The crude was purified byprep-TLC (6.6% methanol in dichloromethane) to afford the title compound(150 mg). MS m/z: 602.3 (M+H)⁺.

Step 2: Intermediate 3

To a solution of Intermediate 2 (150 mg, 0.25 mmol) in DCM (15 mL) wasadded triethylamine (50 mg, 0.49 mmol) and di-tert-butyl dicarbonate (65mg, 0.29 mmol). The resultant mixture was allowed to stir at ambienttemperature for 2 h. The reaction mixture was partitioned between EtOAcand water. The organic phase was separated, washed with 1 N HCl,saturated aqueous NaHCO₃, dried over anhydrous sodium sulfate andevaporated to dryness to afford the title compound (130 mg). MS m/z:702.3 (M+H)⁺.

Step 3: Intermediate 4

To a suspension of Intermediate 3 (130 mg, 0.180 mmol) in ethanol (15mL) and saturated aqueous NH₄Cl (2 mL) was added Fe powder (83.0 mg,1.48 mmol) and the resultant mixture was heated at reflux for 2 h afterwhich it was diluted with DCM (60 mL) the organic phase was separated,dried over anhydrous sodium sulfate, and evaporated to dryness to affordthe titled product (120 mg).

Step 4: Intermediate 5

Intermediate 4 (110 mg, 0.16 mmol), propionic acid (18.0 mg, 0.24 mmol)and HATU (124 mg, 0.33 mmol) were dissolved in DMF (10 mL). The reactionmixture was cooled to 0° C. under N₂ and DIPEA (63 mg, 0.49 mmol) wasadded slowly and the mixture was allowed to stir at ambient temperaturefor 16 h. The reaction mixture was partitioned between EtOAc and water.The organic phase was separated, washed with water, dried over anhydroussodium sulfate and evaporated to dryness. The residue was purified bycolumn chromatography with silica gel (4% MeOH in DCM) to afford thetitle product (80 mg). MS m/z: 728.4 (M+H)⁺

Step 5: I-7

Intermediate 5 (80.0 mg, 0.11 mmol) was dissolved in DCM (2 mL) followedby addition of TFA (5 mL). The resultant mixture was allowed to stir atambient temperature for 1 h after which it was concentrated. The residuewas taken up in DCM, and DIPEA was added until a pH=7 was achieved. Thesolution was cooled to 0° C., acryloyl chloride (15.0 mg, 0.17 mmol) wasadded and the mixture was allowed to stir at 0° C. for 10 min. Thereaction was quenched by addition of saturated aqueous NaHCO₃, extractedwith EtOAc, and the organic layer was dried over anhydrous Na₂SO₄ andconcentrated. The residue was purified by prep-TLC (4% MeOH in DCM) toafford the title compound (20 mg, 26%, two steps). MS m/z: 682.4 (M+H)⁺.¹H NMR (400 MHz, DMSO-d₆): δ: 1.02-1.11 (t, 3H), 1.16-1.30 (m, 3H),1.49-1.70 (m, 3H), 1.76-1.94 (m, 2H), 2.28 (q, 2H), 3.66-3.75 (m, 1H),3.96 (s, 6H), 4.50 (s, 2H), 5.11 (s, 2H), 5.46-5.54 (m, 1H), 6.01 (d,1H), 6.03-6.15 (m, 1H), 6.61-6.70 (m, 1H), 6.99 (s, 1H), 6.94 (d, 1H),7.18 (s, 1H), 7.34-7.43 (m, 1H), 7.57-7.65 (m, 1H), 7.96 (s, 1H), 9.76(s, 1H).

Example 10: Synthesis of I-8, (Racemic)

Step 1: Intermediate 2

To a solution of Intermediate 8 from Example 2 (150 mg, 0.27 mmol) in1,4-dioxane (8 mL) was added cis-cyclohexane-1,2-diamine (93.0 mg, 0.82mmol) and p-TSA (23.4 mg, 0.14 mmol). The reaction mixture was heated at100° C. for 16 h. Water was added and the resultant mixture wasextracted with EtOAc (50 mL×3). The combined organic layers were driedover Na₂SO₄, filtered and concentrated in vacuo to afford the titlecompound (164 mg) which was used without further purification. MS m/z:602.3 [M+1]⁺.

Step 2: Intermediate 3

To a solution of Intermediate 2 (164 mg, 0.27 mmol) in DCM (6 mL) wasadded triethylamine (82.6 mg, 0.82 mmol) and di-tert-butyl dicarbonate(89.0 mg, 0.41 mmol). The reaction mixture was allowed to stir atambient temperature for 2 h. The reaction mixture was concentrated andthe crude product was subjected to column chromatography on silica gel(2% MeOH/DCM) to afford the titled compound (159 mg). ¹H NMR (400 MHz,DMSO-d6): δ 1.34 (s, 9H), 1.42-1.52 (m, 2H), 1.56-1.77 (m, 2H), 3.25 (d,2H), 3.48-3.64 (m, 1H), 3.75-3.91 (m, 1H), 3.97 (s, 6H), 4.55 (s, 2H),5.25 (s, 2H), 6.31-6.47 (m, 1H), 6.54-6.64 (m, 1H), 7.01 (s, 1H), 7.62(t, 1H), 7.76 (d, 1H), 8.02 (s, 1H), 8.07-8.18 (m, 2H).

Step 3: Intermediate 4

To a solution of Intermediate 3 (153 mg, 0.22 mmol) in EtOH (10 mL) andwater (5 mL) was added Fe power (72.0 mg, 1.31 mmol) and NH₄Cl (70.0 mg,1.31 mmol). The mixture was heated at reflux for 2 h. The reactionmixture was filtered and the filtrate was concentrated in vacuo. To theresidue was added water and the aqueous solution extracted with EtOAc(50 mL×3). The combined organic layers were washed with saturatedNaHCO₃, dried over Na₂SO₄, filtered and concentrated in vacuo to affordthe titled compound (96.7 mg) which was used without purification. MSm/z: 672.4 (M+H)⁺

Step 4: Intermediate 5

To a solution of Intermediate 4 (96.7 mg, 0.14 mmol) in DMF (6 mL) wasadded propionic acid (16.0 mg, 0.22 mmol) and HATU (109.5 mg, 0.29mmol). The mixture was cooled to 0° C. and DIPEA (55.7 mg, 0.43 mmol)was added. The reaction mixture was allowed to stir at ambienttemperature for 16 h. Water was added and the mixture was extracted withEtOAc (50 mL×3). The combined organic layers were washed with water,dried (Na₂SO₄), and concentrated in vacuo. The crude product wassubjected to column chromatography on silica gel (3% MeOH/DCM) to affordthe titled compound (98.0 mg). MS m/z: 728.5 (M+H)⁺

Step 5: Intermediate 6

To a solution of Intermediate 5 (98.0 mg, 0.13 mmol) in DCM (2 mL) wasadded TFA (2 mL) and the reaction mixture was allowed to stir at ambienttemperature for 30 min. The reaction mixture was concentrated in vacuo,water was added and the resultant mixture was extracted with EtOAc (50mL×3). The combined organic layers were washed with saturated aqueousNaHCO₃, dried (Na₂SO₄), and concentrated in vacuo to afford the titlecompound (88.3 mg) which was used without further purification. MS m/z:628.4 (M+H)⁺

Step 6: I-8

To a cooled (0° C.) solution of Intermediate 6 (88.3 mg, 0.14 mmol) andDIPEA (36.1 mg, 0.28 mmol) in 4 mL of THF was added acryloyl chloride(15.3 mg, 0.17 mmol). The reaction was allowed to stir at 0° C. for 10min. The mixture was quenched with saturated aqueous NaHCO₃ andextracted with EtOAc (40 mL×3). The combined organic layers were washedwith saturated aqueous NaHCO₃, dried (Na₂SO₄), and concentrated invacuo. The resultant residue was subjected to column chromatography onsilica gel (5% MeOH/DCM) to afford the title compound (55.9 mg) MS m/z:682.3 (M+H)⁺. ¹H NMR (400 MHz, DMSO-d6): δ 1.07 (t, 3H), 1.22-1.83 (m,8H), 2.28 (q, 2H), 3.96 (s, 6H), 4.13 (br, 1H), 4.51 (s, 2H), 5.04-5.14(m, 2H), 5.55 (dd, 2.4 Hz, 1H), 5.74 (s, 1H), 6.05 (dd, 1H), 6.22-6.41(m, 1H), 6.60 (br s, 1H), 6.91-7.05 (m, 2H), 7.18 (t, 1H), 7.41 (d, 1H),7.58 (s, 1H), 7.67 (d, 1H), 7.98 (s, 1H), 9.73 (s, 1H).

Example 11: Synthesis of I-9

The title compound was prepared as described in Example 17 (below) using1-(2-methoxyethyl)piperazine in place of N-ethylpiperidine in Step 2 andusing an intermediate prepared in a manner as described for Intermediate6 in Example 2, which was oxidized using mCPBA as described in Example2. MS m/z: 750.3 (M+H⁺). ¹H NMR (400 MHz, CD₃OD): δ: 7.97 (1H, s), 7.63(1H, s), 7.40 (2H, d), 7.33 (2H, m), 7.16 (1H, t), 6.87 (2H, m), 6.57(2H, s), 6.45 (1H, dd), 6.39 (1H, m), 5.75 (1H, dd), 5.13 (2H, s), 4.75(2H, s), 3.77 (6H, s), 3.76 (1H, m), 3.43 (3H, s), 2.36 (2H, q), 1.62(3H, t)

Example 12: Synthesis of I-10

Compound I-10 was prepared as described in Example 5 using tert-butyl(2-amino-5-(trifluoromethyl)phenyl)carbamate instead ofbenzene-1,2-diamine in Step 1. MS m/z: 673.1 (M+H⁺).

Example 13: Synthesis of I-11

Compound I-11 was prepared as described in Example 5 using4,5-dichlorobenzene-1,2-diamine instead of benzene-1,2-diamine inStep 1. MS m/z: 673.1 (M+H⁺).

Example 14: Synthesis of I-12

Compound I-12 was prepared as described in Example 5 using(3-fluorophenyl)methanamine in place of methylamine in Step 1. MS m/z:623.3 (M+H⁺).

Example 15: Synthesis of I-13

The title compound was prepared as outlined in Example 17 (below) using2-methoxy-N-methylethanamine in place of N-ethylpiperidine in Step 2 andusing a derivative of Intermediate 5 from Example 1 (prepared usingbenzylamine in place of methylamine in Step 5) in place of Intermediate4 in Step 4. MS m/z: 624.3 (M+H⁺). ¹H NMR (400 MHz, CDCl₃): δ: 7.92 (1H,s), 7.29 (1H, d), 7.21 (6H, m), 6.78 (1H, dd), 6.55 (2H, s), 6.46 (1H,s), 6.41 (2H, m), 5.77 (1H, dd), 5.15 (2H, s), 4.51 (2H, s), 3.78 (6H,s), 3.60 (2H, t), 3.54 (2H, t), 3.06 (3H, t).

Example 16: Synthesis of I-14

Compound I-14 was prepared as described in Example 17 using2-methoxy-1-ethanol in place of N-ethylpiperidine in Step 2 and using aderivative of Intermediate 5 from Example 1 (prepared using benzylaminein place of methylamine in Step 5) in place of Intermediate 4 in Step 4.MS m/z: 611.3 (M+H⁺).

Example 17: Synthesis of I-15

Step 1: Intermediate 2

To a solution of Intermediate 1 (2.34 g, 15.0 mmol) in DMF (50 mL) wasadded (BOC)₂O (6.60 g, 30.3 mmol) and DMAP (600 mg, 4.9 mmol). Thereaction was stirred at RT overnight. After removing DMF under reducedpressure, the product was isolated by silica gel chromatography. MS m/z:357.2 (M+H⁺).

Step 2

Intermediate 2 (300 mg, 084 mmol) and N-ethylpiperidine (0.30 mL, 2.19mmol) were mixed in DMF (3.0 mL). The mixture was heated at 110° C. for3.0 h. Then, the reaction was concentrated in vacuo and purified byflash chromatography on silica gel to afford 330 mg of the titlecompound. MS m/z: 351.3 (M+H)⁺.

Step 3: Intermediate 3

The product from Step 2 was dissolved in MeOH (10 mL), and 10 wt % Pd/C(100 mg) was added. The reaction mixture was allowed to stir at ambienttemperature under an H₂ balloon for 4.5 hr. The reaction mixture wasfiltered through a short plug of celite and concentrated in vacuo toafford 275 mg of the title compound which was used without purification.MS m/z: 292.1 (M+H⁺).

Step 4: Intermediate 5

Intermediate 3 (200 mg, 0.68 mmol), Intermediate 6 (from Example 1) (150mg, 0.37 mmol), and TFA (5.0 μL) were taken up in 1,4-dioxane (2.0 mL)and the reaction mixture was heated at 110° C. for 16 h. The reactionmixture was allowed to cool to ambient temperature, concentrated andpurified by silica gel chromatography to afford 110 mg of the titlecompound. MS m/z: 619.8 (M+H)⁺.

Step 4: I-15

Intermediate 5 was dissolved in 20% TFA/DCM at ambient temperature andallowed to stir for 3.5 h. The reaction mixture was concentrated, theresultant residue was taken up in DCM and treated with silica supportedcarbonate and the mixture filtered. The filtrate was concentrated thendissolved in THF (2.0 mL) and cooled to −10° C., followed by addition ofacryloyl chloride (7.0 μL, 0.086 mmol). After 10 min, the reaction wasconcentrated and purified by prep-HPLC. MS m/z: 573.2 (M+H⁺). ¹H NMR(400 MHz, CDCl₃): δ: 8.67 (1H, s), 7.78 (1H, s), 7.67 (1H, s), 7.48 (1H,d), 6.67 (1H, dd), 6.40 (5H, m), 5.74 (1H, dd), 4.63 (2H, s), 3.77 (6H,s), 3.66 (4H, m), 3.41 (2H, m), 3.33 (3H, s), 3.14 (2H, m), 2.89 (2H,m), 1.39 (3H, t).

Example 18: Synthesis of I-16

Compound I-16 was prepared as described in Example 5, using(4-methoxyphenyl)methanamine in place of methylamine to prepare thestarting material. MS m/z: 635.4 (M+H⁺).

Example 19: Synthesis of I-17

The title compound was prepared as described in Example 17 usingmorpholine in place of N-ethylpiperidine in Step 2 and Intermediate 5from Example 1 in place of Intermediate 6 in Step 4. MS m/z: 546.2(M+H⁺). ¹H NMR (400 MHz, CD₃OD): δ: 7.87 (1H, s), 7.40 (1H, d), 7.25(1H, s), 6.98 (1H, dd), 6.54 (2H, s), 6.46 (1H, m), 6.35 (1H, dd), 5.80(1H, dd), 4.71 (2H, s), 3.84 (4H, t), 3.77 (6H, s), 3.38 (3H, s), 3.21(4H, t).

Example 20: Synthesis of I-18 (Racemic)

Compound I-18 was prepared as described in Example 21 using mCPBAinstead of SO₂Cl₂ in Step 1 (mCPBA oxidation is described in Example 2,Step 7). MS m/z: 476.4 (M+H⁺).

Example 21: Synthesis of I-19, (Racemic)

Step 1: Intermediate 2

To an ice-cooled solution of Intermediate 1, prepared as described inExample 2 using methylamine in place of 3-nitrobenzylamine in Step 2 andskipping Step 6 (250 mg, 0.72 mmol), in MeCN (2 mL) and DCM (4 mL) wasadded sulfuryl chloride (0.12 mL, 1.44 mmol). The reaction was stirredat 0° C. for 15 min. The reaction was quenched with saturated aqueousNaHCO₃ and the aqueous layer was extracted with DCM (30 mL×3). Thecombined organic layers were dried (Na₂SO₄), filtered and concentratedin vacuo. The crude product was purified by column chromatography onsilica gel (2% MeOH/DCM) to afford 192 mg, of the title compound. ¹H NMR(400 MHz, DMSO-d6): δ 2.96 (s, 3H), 3.41 (s, 3H), 4.03 (s, 6H), 4.81 (s,2H), 7.08 (s, 1H), 8.64 (s, 1H).

Step 2: Intermediate 3

To a solution of Intermediate 2 (60.0 mg, 0.14 mmol) in 1,4-dioxane (5mL) was added cis-cyclohexane-1,2-diamine (47.7 mg, 0.42 mmol) and p-TSA(12.0 mg, 0.07 mmol). The reaction was heated at 105° C. overnight. Themixture was cooled to ambient temperature, water was added, and theresulant mixture was extracted with EtOAc (50 mL×3). The combinedorganic layers were washed with saturated aqueous NaHCO₃, dried(Na₂SO₄), filtered and concentrated in vacuo. The crude product waspurified by column chromatography on silica gel (10% MeOH/DCM) to afford73.9 mg, of the title compound. MS m/z: 481.3 (M+1)⁺

Step 3: I-19

To an ice-cooled solution of Intermediate 3 (73.9 mg, 0.15 mmol) andDIPEA (39.7 mg, 0.31 mmol) in THF (3 mL) was added acryloyl chloride(16.7 mg, 0.18 mmol). The reaction was allowed to stir at 0° C. for 10min. The reaction was quenched with saturated aqueous NaHCO₃ andextracted with EtOAc (30 mL×3). The combined organic layers were washedwith saturated aqueous NaHCO₃, dried (Na₂SO₄), filtered and concentratedin vacuo. The crude product was purified by column chromatography onsilica gel (5% MeOH/DCM) to afford 40.7 mg of the title compound. MSm/z: 535.3 (M+1)⁺. ¹H NMR (400 MHz, DMSO-d6): δ1.35-1.84 (m, 8H), 3.96(s, 6H), 4.11-4.29 (m, 2H), 4.44 (s, 2H), 5.50-5.75 (m, 1H), 5.99-6.25(m, 1H), 6.23-6.47 (m, 1H), 6.52-6.86 (m, 1H), 6.97-7.18 (m, 1H),7.66-7.90 (m, 1H), 7.96-8.14 (m, 1H).

Example 22: Synthesis of I-20

The title compound was prepared as described in Example 17 using1-(2-methoxyethyl)piperazine in place of N-ethylpiperidine in Step 2 andIntermediate 5 (from Example 1) in place of Intermediate 6 in Step 4. MSm/z: 603.3 (M+H⁺). ¹H NMR (400 MHz, CD₃OD): δ: 7.94 (1H, s), 7.50 (1H,d), 7.40 (1H, d), 7.00 (1H, dd), 6.54 (2H, s), 6.44 (2H, m), 6.39 (1H,m), 5.80 (1H, dd), 4.71 (2H, s), 3.79 (6H, s), 3.44 (5H, m), 3.31 (3H,s).

Example 23: Synthesis of I-21

The title compound was prepared as described in Example 17 using2-methoxy-N-methylethanamine in place of N-ethylpiperidine in Step 2 andIntermediate 5 (from Example 1) in place of Intermediate 6 in Step 4. MSm/z: 548.3 (M+H⁺). ¹H NMR (400 MHz, CD₃OD): δ: 7.86 (1H, s), 7.39 (1H,d), 7.15 (1H, s), 6.85 (1H, dd), 6.53 (2H, m), 6.46 (1H, m), 6.35 (1H,m), 5.79 (1H, dd), 4.70 (2H, s), 3.77 (6H, s), 3.59 (2H, s), 3.38 (3H,s), 3.06 (3H, s).

Example 24: Synthesis of I-22 (Racemic)

Compound I-22 was prepared as described in Example 21 usingtrans-cyclohexane-1,2-diamine in place of cis-cyclohexane-1,2-diamine inStep 2. MS m/z: 535.3 (M+H⁺).

Example 25: Synthesis of I-23

Step 1: Intermediate 2

A solution of Intermediate 1 (750 mg, 3.93 mmol) in cyclopropanamine (5mL) was stirred at ambient temperature overnight after which thesolution was concentrated, triturated with EtOAc and filtered to afford700 mg of the title compound. MS m/z: 212.1 (M+H)⁺

Step 2: Intermediate 3

To a solution of Intermediate 2 (700 mg, 3.32 mmol) in DCM (30 mL) wasadded manganese oxide (2.30 g, 26.4 mmol) and the mixture was allowed tostir at ambient temperature for 3 h. The reaction mixture was filteredthrough Celite and washed with DCM and the filtrate was concentrated toafford 550 mg of the title compound. MS m/z: 210.2 (M+H)⁺

Step 3: Intermediate 4

To a solution of Intermediate 3 (550 mg, 2.63 mmol) in DCM (20 mL) wasadded 3, 5-dimethoxyaniline (480 mg, 3.14 mmol) and acetic acid (0.13 g,2.17 mmol) under nitrogen. The mixture was stirred at ambienttemperature for 15 min followed by the addition of NaBH(OAc)₃ in twoportions (0.55 g×2, 5.19 mmol) at a 15 min interval. The reaction wasallowed to stir at ambient temperature for 17 h after which it wasquenched with 1M NaOH and allowed to stir for 15 min. The aqueous layerwas extracted with DCM (50 mL×3) and the combined organic layers werewashed with brine, dried (Na₂SO₄), filtered and concentrated. The crudeproduct was subjected to flash chromatography on silica gel (10% EtOAcin DCM) to afford 0.83 g of the title compound. MS m/z: 347.2 (M+H)⁺

Step 4: Intermediate 5

To a solution of Intermediate 4 (830 mg, 2.41 mmol) in THF (20 mL) wasadded triphosgene (890 mg, 3.57 mmol) followed by addition oftriethylamine (1.20 g, 11.9 mmol) under nitrogen. The mixture wasallowed to stir at ambient temperature for 1 h, then at reflux for 3 h.The reaction was quenched with a 1:1 mixture of saturated aqueousNaHCO₃/H₂O (20 mL) and extracted with EtOAc (60 mL×3). The combinedorganic layers were washed with brine, dried (Na₂SO₄), filtered andconcentrated. The crude product was washed with hexanes to afford 500 mgof the title compound. MS m/z: 373.3 (M+H)⁺

Step 5: Intermediate 6

To an ice-cooled solution of Intermediate 5 (150 mg, 0.40 mmol) in MeCN(6 mL) and DCM (12 mL) was added sulfuryl chloride (108 mg, 0.80 mmol).The reaction was allowed to stir at 0° C. for 10 min. The reaction wasquenched with saturated aqueous NaHCO₃ and the aqueous was extractedwith DCM (50 mL×3). The combined organic layers were dried (Na₂SO₄),filtered and concentrated. The crude was purified by prep-TLC (5% MeOHin DCM) to afford 60 mg of the title compound. MS m/z: 457.2 (M+H)⁺. ¹HNMR (400 MHz, DMSO-d6): δ 0.62-0.70 (m, 2H), 1.09-1.00 (m, 2H),2.73-2.83 (m, 1H), 2.91 (s, 3H), 3.97 (s, 6H), 4.60-4.69 (m, 2H), 7.01(s, 1H), 8.62 (s, 1H)

Step 6: Intermediate 7

To a solution of Intermediate 6 (60 mg, 0.13 mmol) in dioxane (10 mL)was added benzene-1,2-diamine (43 mg, 0.39 mmol) and catalytic p-TSA (2mg). The resultant mixture was heated to reflux overnight. The reactionmixture was cooled to ambient temperature, diluted with EtOAc, washedwith saturated aqueous NaHCO₃, dried over Na₂SO₄ and concentrated. Thecrude product was purified by prep-TLC (2% MeOH in DCM) to afford 30 mgof the title compound. MS m/z: 501.3 (M+H)⁺. ¹H NMR (400 MHz, DMSO-d6):δ 0.62 (d, 2H), 0.95 (d, 2H), 2.60 (dd, 1H), 3.95 (s, 6H), 4.39 (s, 2H),4.83 (s, 2H), 6.47-6.63 (m, 1H), 6.73 (dd, 1H), 6.82-6.86 (m, 1H), 6.98(s, 1H), 8.06 (s, 1H), 7.51 (d, 1H), 8.39 (s, 1H),

Step 7: I-23

A solution of Intermediate 7 (30 mg, 0.06 mmol) in THF (10 mL) was addedDIPEA (15 mg, 0.12 mmol) and cooled to −78° C. To the reaction mixturewas added acryloyl chloride (6.5 mg, 0.07 mmol). The reaction mixturewas stirred at -78° C. for 10 min. after which it was quenched with asaturated aqueous NaHCO₃ solution, extracted with EtOAc, andconcentrated. The crude was purified by prep-TLC (3˜4% MeOH in DCM) toafford 20 mg of the title compound. MS m/z: 555.3 (M+H)⁺. ¹H NMR (400MHz, DMSO-d₆): δ 0.55-0.67 (m, 2H), 0.96 (q, 2H), 2.61-2.64 (m, 1H),3.95 (s, 6H), 4.41 (s, 2H), 5.78 (dd, 1H), 6.28 (dd, 1H), 6.52 (dd, 1H),6.98 (s, 1H), 7.06-7.12 (m, 1H), 7.17-7.24 (m, 1H), 7.51 (d, 1H), 7.98(d, 1H), 8.11 (s, 1H), 8.53 (s, 1H), 9.83 (s, 1H).

Example 26: Synthesis of I-24

Compound I-24 was prepared as described in Example 25 using(4-chloro-3-methoxyphenyl)methanamine in place of cyclopropanamine inStep 2. MS m/z: 669.4 (M+H⁺).

Example 27: Synthesis of I-25

Compound I-25 was prepared as described in Example 25 using(4-fluorophenyl)methanamine in place of cyclopropanamine in Step 2. MSm/z: 623.3 (M+H⁺).

Example 28: Synthesis of I-26

Compound I-26 was prepared as described in various steps from Example 3but in the following order: Step 1, Step 6, Step 3. MS m/z: 620.4(M+H⁺).

Example 29: Synthesis of I-27

Step: Intermediate 2

To a solution of Intermediate 1 (466 mg, 2.15 mmol) in DMF (6.0 mL) wasadded (BOC)₂O (515 mg, 2.36 mmol) and DMAP (20 mg). The reaction wasstirred at ambient temperature for 16 h. DMF was removed under reducedpressure and purified by silica gel chromatography to afford 480 mg ofthe title compound. MS m/z: 317.1 (M+H⁺).

Step 2 Intermediate 4

Intermediate 2 (90 mg, 0.28 mmol), Intermediate 3 (120 mg, 0.58 mmol),and Pd(dppf)Cl₂ (22.0 mg, 0.03 mmol) were combined in 1,4-dioxane (5 mL)and 2 M aqueous Na₂CO₃ (1.2 mL). The mixture was heated at 110° C. for30 min. The reaction mixture was diluted with EtOAc, washed withsaturated aqueous NaHCO₃ and brine, dried (Na₂SO₄), filtered andconcentrated in vacuo. The residue was subjected to flash chromatographyon silica gel to afford 47 mg of the title compound. MS m/z: 319.2(M+H⁺).

Step 3 Intermediate 4

Intermediate 4 (47.0 mg, 0.15 mmol) was dissolved in MeOH (5 mL), towhich was added 10 wt % Pd/C (20 mg). The reaction mixture was allowedto stir at ambient temperature under a H₂ balloon for 3.5 hr. Thereaction was filtered through a short plug of celite and concentrated invacuo to afford the title compound (quantitative) which was useddirectly. MS m/z: 288.1 (M+H⁺).

I-27

The title compound was prepared as outlined in Example 5 using commonIntermediate 5 from Example 1. MS m/z: 541.2 (M+H⁺). ¹H NMR (400 MHz,CD₃OD): δ: 7.92 (1H, s), 7.31 (2H, m), 7.18 (2H, m), 6.92 (1H, m), 6.86(1H, s), 6.53 (2H, m), 6.46 (1H, dd), 5.44 (1H, dd), 5.01 (2H, s), 4.89(6H, s), 4.64 (1H, m), 4.57 (1H, s), 3.93 (3H, s), 3.87 (2H, m), 3.29(1H, m).

Example 30: Synthesis of I-28

Compound I-28 was prepared as described in Example 5. The startingmaterial was prepared using (3-methoxyphenyl)methanamine in place ofmethylamine. MS m/z: 654.5 (M+H⁺).

Example 31: Synthesis of I-29

Step 1: Intermediate 2

A solution of Intermediate 1 (3.0 g, 21.3 mmol) in methanamine (ethanolsolution, 30 mL) was allowed to stir at ambient temperature for 2 hafter which the volatiles were evaporated under reduced pressure. To theresidue was added aqueous solution of NaHCO₃ (50 mL) and the mixture wasextracted with EtOAc (30 mL×3). The combined organic layers were driedover Na₂SO₄ and concentrated to give 3.53 g of the title compound. ¹HNMR (400 MHz, CDCl₃): δ 3.03 (d, 3H), 6.63-6.67 (m, 1H), 6.84 (d, 1H),7.43-7.48 (m, 1H), 7.99 (br s, 1H), 8.17 (dd, 1H).

Step 2: Intermediate 3

To a solution of Intermediate 3 (500 mg, 3.29 mmol) and DIPEA (900 mg,7.0 mmol) in DMF (5 mL), was added acryloyl chloride (1.92 g, 21.4mmol). The reaction was stirred at ambient temperature for 2.5 h. Themixture was partitioned between EtOAc and water. The organic layer wasseparated and washed with water, brine, dried over Na₂SO₄ and the crudeproduct was subjected to flash chromatography on silica gel (elutingwith 6% MeOH in DCM) to afford 583 mg of the title compound. MS m/z:207.2 (M+1)⁺. ¹H NMR (400 MHz, CDCl₃): δ 3.32 (s, 3H), 5.53 (d, 1H),5.82-5.89 (m, 1H), 6.37 (d, 1H), 7.37 (d, 1H), 7.56 (t, 1H), 7.67-7.71(m, 1H), 8.01 (d, 1H).

Step 3: Intermediate 4

A mixture of Intermediate 3 (200 mg, 0.97 mmol) and Fe (272 mg, 4.85mmol) in aqueous NH₄Cl (3 mL) and EtOH (6 mL) were stirred at 80° C. for1 h. Solids were removed by filtration and the filtrate wasconcentrated. The residue was diluted with water and extracted withEtOAc (20 mL×3), the organic layer was washed with brine, dried (Na₂SO₄)and concentrated in vacuo. The crude product was purified by flashchromatography on silica gel (eluting with 5% MeOH in DCM) to afford 105mg of the title compound. MS m/z: 177.2 (M+1)⁺. ¹H NMR (400 MHz, CDCl₃):δ 3.26 (s, 3H), 3.76 (br, 2H), 5.52 (dd, 1H), 6.07 (dd, 1H), 6.39 (dd,1H), 6.74-6.80 (m, 1H), 6.99 (dd, 1H), 7.14-7.18 (m, 1H), 7.26 (s, 1H)

Step 4: I-29

A mixture of Intermediate 6 from Example 1 (100 mg, 0.24 mmol),Intermediate 4 (44 mg, 0.24 mmol), Cs₂CO₃ (162 mg, 0.48 mmol), Pd₂(dba)₃(21.4 mg, 0.038 mmol), and Xantphos (43.0 mg, 0.076 mmol) in 1,4-dioxane(2 mL) were allowed to stir at 95° C. under N₂ for 5 h. The volatileswere evaporated under reduced pressure. The residue was partitionedbetween EtOAc and water and the organic layer was washed with brine,dried (Na₂SO₄), filtered and concentrated in vacuo. The crude productwas subjected to flash chromatography on silica gel (eluting with 50%EtOAc in hexanes) to afford 29.8 mg mg of the title compound. MS m/z:543.3 (M+1)⁺. ¹H NMR (400 MHz, DMSO-d6): δ 3.15 (s, 3H), 3.25 (s, 3H),4.00 (s, 6H), 4.52 (s, 2H), 5.34-5.57 (m, 1H), 5.88-6.16 (m, 2H), 7.00(s, 1H), 7.17-7.23 (m, 2H), 7.34-7.43 (m, 1H), 7.87 (d, 1H), 8.05 (s,1H), 8.76 (s, 1H).

Example 32: Synthesis of I-30

Compound I-30 was prepared as described in Example 17 using Intermediate5 (from Example 1), in place of Intermediate 6. MS m/z: 573.5 (M+H⁺).

Example 33: Synthesis of I-31

Steps 1: Intermediate 2

To a solution of Intermediate 1 (2.34 g, 15.0 mmol) in DMF (50 mL) wasadded (BOC)₂O (6.60 g, 30.3 mmol) and DMAP (600 mg, 4.9 mmol). Thereaction was allowed to stir at ambient temperature overnight. DMF wasremoved under reduced pressure and the title compound was isolated bysilica gel chromatography. MS m/z: 257.2 (M+H⁺).

Step 2: Intermediate 3

Intermediate 2 (660 mg, 2.57 mmol) and piperidine (0.65 mL, 6.60 mmol)were combined in DMF (6.0 mL). The reaction mixture was heated at 110°C. for 3 h. The reaction was concentrated in vacuo and purified by flashchromatography on silica gel to afford 660 mg of the title compound. MSm/z: 322.3 (M+H⁺).

Step 3: Intermediate 4

Intermediate 3 (660 mg, 2.06 mmol) was dissolved in MeOH (10 mL), towhich was added 10 wt % Pd/C (100 mg). The reaction was allowed to stirunder H₂ balloon for 4.5 hr. The reaction was filtered through a shortplug of celite and concentrated in vacuo. Without purification, thecrude product (quant.) was used directly for the next step. MS m/z:292.1 (M+H)⁺.

I-31

The title compound was prepared from Intermediate 4 and Intermediate 5(from Example 1) using the procedure as described in Example 5. MS m/z:544.3 (M+H⁺). ¹H NMR (400 MHz, CDCl₃): δ: 8.65 (1H, s), 8.24 (1H, s),7.71 (1H, m), 7.44 (1H, m), 6.41 (5H, m), 5.79 (1H, dd), 4.65 (2H, s),3.77 (6H, s), 3.39 (4H, m), 3.34 (3H, s), 2.00 (4H, m), 1.67 (2H, m).

Example 34: Synthesis of I-32

Compound I-32 was prepared as described in Example 25 using allylaminein place of cyclopropanamine in Step 1 and mCPBA instead of SO₂Cl₂ inStep 5 (mCPBA oxidation is described in Example 2, step 7). MS m/z:487.1 (M+H⁺).

Example 35: Synthesis of I-33

Compound I-33 was prepared as described in Example 25 using3-aminopropane-1,2-diol in place of cyclopropanamine in Step 1 and mCPBAinstead of SO₂Cl₂ in Step 5 (mCPBA oxidation is described in Example 2,step 7). MS m/z: 521.2 (M+H⁺).

Example 36: Synthesis of I-34

Compound I-34 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using cyclopropanaminein place of methylamine in Step 5. MS m/z: 487.3 (M+H⁺).

Example 37: Synthesis of I-35 (Racemic)

Compound I-35 was prepared as described in Example 21. The startingmaterial was prepared as described in Example 2 usingpyridin-3-ylmethanamine in place of 3-nitrobenzylamine in Step 2. MSm/z: 612.3 (M+H⁺).

Example 38: Synthesis of I-36

Compound I-36 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 2 using tert-butyl3-aminopiperidine-1-carboxylate in place of 3-nitrobenzyl amine in Step2 and skipping Step 6. MS m/z: 630.3 (M+H⁺).

Example 39: Synthesis of I-37

Compound I-37 was prepared as described in Example 5 using tert-butyl(2-aminophenyl)(methyl)carbamate in place of 1,2-benzenediamine inStep 1. A BOC deprotection step was performed prior to Step 2 (asdescribed in Example 3, Step 5). MS m/z: 619.4 (M+H⁺).

Example 40: Synthesis of I-38

Compound I-38 was prepared as described in Example 53 using aniline inplace of cyclopropanamine in Step 1. MS m/z: 401.3 (M+H⁺).

Example 41: Synthesis of I-39 (Racemic)

Compound I-39 was prepared as described in Example 21 using mCPBAinstead of SO₂Cl₂ in Step 1 (mCPBA oxidation is described in Example 2,Step 7) and tert-butyl (cis-2-aminocyclohexyl)(methyl)carbamate in placeof cis-cyclohexane-1,2-diamine in Step 2. A BOC deprotection step wasperformed prior to Step 3 (as described in Example 3, Step 5). MS m/z:481.5 (M+H⁺).

Example 42: Synthesis of I-40

The title compound was prepared as described in Example 7. The startingmaterial was prepared as described in Example 2 using tert-butyl4-aminopiperidine-1-carboxylate in place of 3-nitrobenzyl amine in Step2 and skipping Step 6. A final BOC deprotection step was performed asdescribed in Example 3, Step 5. MS m/z: 544.3 (M+H⁺). ¹H NMR (400 MHz,CD₃OD): δ: 8.02 (1H, s), 7.76 (1H, d), 7.55 (1H, d), 7.36 (2H, m), 6.53(2H, s), 6.46 (1H, m), 6.36 (1H, dd), 5.80 (1H, dd), 4.73 (2H, s), 3.82(2H, d), 3.77 (6H, s), 3.30 (2H, s), 2.81 (2H, t), 1.92 (1H, m), 1.68(2H, m), 1.30 (2H, q).

Example 43: Synthesis of I-41

Compound I-41 was prepared as described in Example 7 using tert-butyl(2-amino-5-methylphenyl)carbamate in place of 1,2-benzenediamine inStep 1. A BOC deprotection step was performed prior to Step 2 (asdescribed in Example 3, Step 5). MS m/z: 475.4 (M+H⁺).

Example 44: Synthesis of I-42

Compound I-42 was prepared as described in Example 7 using tert-butyl(2-amino-5-trifluoromethylphenyl)carbamate in place of1,2-benzenediamine in Step 1. A BOC deprotection step was performedprior to Step 2 (as described in Example 3, Step 5). MS m/z: 529.4(M+H⁺).

Example 45: Synthesis of I-43

The title compound was prepared as described in Example 7. The startingmaterial was prepared as described in Example 2 using tert-butyl3-aminopiperidine-1-carboxylate in place of 3-nitrobenzyl amine in Step2, and skipping Step 6. A final BOC deprotection step was performed asdescribed in Example 3, Step 5. MS m/z: 530.2 (M+H⁺). ¹H NMR (400 MHz,CDCl₃): δ: 7.87 (1H, s), 7.68 (1H, m), 7.43 (1H, m), 7.24 (1H, m), 7.18(2H, m), 6.32 (4H, m), 5.68 (1H, dd), 4.85 (1H, m), 4.52 (2H, s), 4.13(1H, m), 3.70 (6H, s), 3.30 (3H, m), 2.39 (2H, m), 1.72 (2H, m), 1.88(2H, m).

Example 46: Synthesis of I-44

The title compound was prepared as outlined in Example 5. Intermediate 1was prepared as described in Example 1 using pyridin-3-ylmethanamine inplace of methylamine in Step 5. MS m/z: 538.3 (M+H⁺). ¹H NMR (400 MHz,CD₃OD): δ: 8.62 (2H, m), 8.23 (1H, d), 8.05 (1H, s), 7.78 (1H, m), 7.65(1H, dd), 7.48 (1H, dd), 7.31 (2H, m), 6.55 (2H, s), 6.46 (1H, s), 6.36(2H, m), 5.77 (1H, dd), 5.22 (2H, dd), 4.75 (2H, s), 3.77 (6H, s).

Example 47: Synthesis of I-45 (Racemic)

Compound I-45 was prepared as described in Example 10. The startingmaterial was prepared as described in Example 2 using tert-butyl3-aminopiperidine-1-carboxylate in place of 3-nitrobenzyl amine in Step2 and skipping Step 6. MS m/z: 636.3 (M+H⁺).

Example 48: I-46 (Racemic)

The title compound was prepared as described in Example 21 using mCPBAinstead of SO₂Cl₂ in Step 1 (mCPBA oxidation is described in Example 2,Step 7). The starting material was prepared as described in Example 2using tert-butyl 3-aminopiperidine-1-carboxylate in place of3-nitrobenzyl amine in Step 2 and skipping Step 6. A final BOCdeprotection step was performed as described in Example 3, Step 5. MSm/z: 536.3 (M+H⁺). ¹H NMR (400 MHz, CD₃OD): δ: 7.98 (1H, s), 6.51 (2H,s), 6.46 (1H, s), 6.17 (2H, m), 5.62 (1H, dd), 5.03 (1H, m), 4.62 (2H,m), 4.28 (1H, m), 3.93 (1H, m), 3.77 (6H, s), 3.40 (2H, m), 2.94 (1H,m), 2.59 (1H, m), 2.10 (2H, m), 1.96 (2H, m), 1.77 (6H, m), 1.54 (2H,m).

Example 49: I-47, (Racemic)

The title compound was prepared as described in Example 21. The startingmaterial was prepared as described in Example 2 using2-aminoacetonitrile in place of 3-nitrobenzy 1 amine in Step 2. MS m/z:560.2 (M+H⁺). ¹H NMR (400 MHz, CD₃OD): δ: 8.07 (1H, s), 6.92 (1H, s),6.33 (1H, m), 6.18 (1H, dd), 5.63 (1H, dd), 5.01 (2H, s), 4.61 (2H, s),4.48 (2H, m), 3.97 (6H, s), 1.79 (6H, m), 1.55 (2H, m).

Example 50: I-48

The title compound was prepared as described in Example 4. The startingmaterial was prepared as described in Example 2 using2-aminoacetonitrile in place of 3-nitrobenzylamine in Step 2, andskipping Step 6. MS m/z: 486.2 (M+H⁺). ¹H NMR (400 MHz, CD₃OD): δ: 8.06(1H, s), 7.76 (1H, d), 7.51 (1H, d), 7.32 (2H, m), 6.56 (2H, s), 6.47(3H, m), 5.79 (1H, dd), 4.7 (2H, s), 4.72 (2H, s), 3.77 (6H, s).

Example 51: Synthesis of I-49

Compound I-49 was prepared as described in Example 7 using tert-butyl(2-amino-5-methylphenyl)carbamate in place of 1,2-benzenediamine inStep 1. A BOC deprotection step was performed prior to Step 2 (asdescribed in Example 3, Step 5). MS m/z: 475.4 (M+H⁺).

Example 52: Synthesis of I-50

Compound I-50 was prepared as described in Example 7 using tert-butyl(2-amino-5-trifluoromethylphenyl)carbamate in place of1,2-benzenediamine in Step 1. A BOC deprotection step was performedprior to Step 2 (as described in Example 3, Step 5). MS m/z: 529.4(M+H⁺).

Example 53: I-51

Step 1: Intermediate 1

To a solution of 2,4-dichloro-5-(iodomethyl)pyrimidine (1.70 g, 5.88mmol) in MeCN (5 mL) and toluene (15 mL) was add aqueous NaOH (1.5 mL5.88 mmol), cyclopropanamine (580 mg, 5.88 mmol) in MeCN (6 mL)/toluene(6 mL). The mixture was allowed to stir at 0° C. for 4 h. The mixturewas partitioned between EtOAc and H₂O. The organic layer was washed withbrine, dried over Na₂SO₄ and concentrated. The residue was purified bycolumn chromatography on silica gel (eluting with 30% EtOAc in hexanes)to afford 540 mg of the title compound. MS m/z: 218.2 (M+1)⁺. ¹H NMR(400 MHz, CDCl₃): δ 0.40-0.54 (m, 4H), 2.13-2.18 (m, 1H), 3.97 (s, 2H),8.62 (s, 1H)

Step 2: Intermediate 2

A solution of Intermediate 1 (540 mg, 2.55 mmol) in methanamine (ethanolsolution, 10 mL) was allowed to stir at 0° C. for 1 h. The volatileswere evaporated under reduced pressure. The residue was partitionedbetween EtOAc and water and the organic layer was washed with brine,dried over Na₂SO₄ and concentrated. The residue was purified by columnchromatography on silica gel (eluting with 5% MeOH in DCM) to afford 350mg of the title compound. MS m/z: 213.3 (M+1)⁺. ¹H NMR (400 MHz, CDCl₃):δ 0.29-0.33 (m, 2H), 0.45-0.50 (m, 2H), 2.08-2.13 (m, 1H), 2.98 (d, 3H),3.74 (s, 2H), 7.17 (br, 1H), 7.75 (s, 1H).

Step 3: Intermediate 3

A mixture of Intermediate 2 (100 mg, 0.47 mmol), triphosgene (83 mg,0.28 mmol), TEA (95 mg, 0.94 mmol) in THF (3 mL) were allowed to stir at80° C. for 16 h. The mixture was partitioned between EtOAc and H₂O. Theorganic layer was washed with brine, dried over Na₂SO₄ and concentrated.The residue was purified by column chromatography on silica gel (elutingwith 2% MeOH in DCM) to afford 60 mg of the title compound. MS m/z:239.3 (M+1)⁺.

Step 4: Intermediate 4

A mixture of Intermediate 3 (60 mg, 0.25 mmol), benzene-1,2-diamine(32.7 mg, 0.30 mmol), Cs₂CO₃ (164 mg, 0.50 mmol), Pd₂(dba)₃ (21.7 mg,0.038 mmol) and Xantphos (43.7 mg, 0.076 mmol) in 1,4-dioxane (2 mL) wasallowed to stir at 90° C. for 16 h under N₂ atmosphere. The volatileswere evaporated under reduced pressure. The residue was diluted withwater and extracted with EtOAc (20 mL×3). The organic layer was washedwith brine, dried over Na₂SO₄, and concentrated. The residue waspurified by column chromatography on silica gel (eluting with 5% MeOH inDCM) to afford 39 mg of the title compound. MS m/z: 311.3 (M+1)⁺

Step 5: I-51

To a solution of Intermediate 4 (39 mg, 0.13 mmol) and DIPEA (38.7 mg,0.3 mmol) in THF (5 mL) at -78° C. was added acryloyl chloride (13.6 g,0.15 mmol). The reaction mixture was allowed to stir at -78° C. for 20min. Water was added and the mixture was extracted with EtOAc (20 mL×3).The organic layer was washed with brine, dried over Na₂SO₄ andconcentrated. The residue was purified by column chromatography onsilica gel (eluting with 5% MeOH in DCM) to afford 9.3 mg of the titlecompound. MS m/z: 365.4 (M+1)⁺. ¹H NMR (400 MHz, CDCl₃): δ 0.63-0.71 (m,2H), 0.88 (q, 2H), 2.62-2.69 (m, 1H), 3.30 (s, 3H), 4.25 (s, 2H), 5.71(d, 1H), 6.21 (dd, 1H), 6.38 (d, 1H), 7.18-7.22 (m, 2H), 7.28 (br s,1H), 7.50 (br, 1H), 7.89 (s, 1H), 8.38 (br s, 1H).

Example 54: Synthesis of I-52

Compound I-52 was prepared as described in Example 53 usingcyclohexanamine in place of cyclopropanamine in Step 1. MS m/z: 407.4(M+H⁺).

Example 55: Synthesis of I-53

Compound I-53 was prepared as described in Example 21 using tert-butyl(cis-2-aminocyclohexyl)(methyl)carbamate in place ofcis-cyclohexane-1,2-diamine in Step 2. A BOC deprotection step wasperformed prior to Step 3 (as described in Example 3, Step 5). MS m/z:549.5 (M+H⁺).

Example 56: Synthesis of I-54

Compound I-54 was prepared as described in Example 17 using tert-butyl(2-amino-5-(1-ethylpiperidin-4-yl)phenyl)carbamate in place ofIntermediate 3. MS m/z: 572.6 (M+H⁺).

Example 57: Synthesis of I-55

Compound I-55 was prepared as described in Example 17 using tert-butyl(5-(4-acetylpiperazin-1-yl)-2-aminophenyl)carbamate in place ofIntermediate 3. MS m/z: 587.5 (M+H⁺).

Example 58: Synthesis of I-56

Compound I-56 was prepared as described in Example 17 using tert-butyl(2-amino-5-(1-ethyl-1,2,3,6-tetrahydropyridin-4-yl)phenyl)carbamate inplace of Intermediate 3. MS m/z: 570.6 (M+H⁺).

Example 59: Synthesis of I-57

Compound I-57 was prepared as described in Example 25 usingbut-3-en-1-amine in place of cyclopropanamine in Step 1 and mCPBAinstead of SO₂Cl₂ in Step 5 (mCPBA oxidation is described in Example 2,step 7). MS m/z: 501.1 (M+H⁺).

Example 60: Synthesis of I-58

Compound I-58 was prepared as described in Example 25 using4-aminobutane-1,2-diol in place of cyclopropanamine in Step 1 and mCPBAinstead of SO₂Cl₂ in Step 5 (mCPBA oxidation is described in Example 2,step 7). MS m/z: 535.1 (M+H⁺).

Example 61: I-59

The title compound was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using2-methoxyethanamine in place of methylamine in Step 5 and skipping Step6. MS m/z: 505.3 (M+H⁺). ¹H NMR (400 MHz, CD₃OD): δ: 7.97 (1H, s), 7.67(1H, m), 7.56 (1H, m), 7.34 (2H, m), 6.53 (2H, s), 6.46 (2H, m), 6.39(1H, m), 5.80 (1H, dd), 4.70 (2H, s), 4.20 (2H, t), 3.77 (6H, s), 3.57(2H, t), 3.25 (3H, s).

Example 62: Synthesis of I-60

Compound I-60 was prepared as described in Example 25 using tert-butyl4-(aminomethyl)piperidine-1-carboxylate in place of cyclopropanamine inStep 1 and mCPBA instead of SO₂Cl₂ in Step 5 (mCPBA oxidation isdescribed in Example 2, step 7). MS m/z: 644.7 (M+H⁺).

Example 63: I-61, (Racemic)

The title compound was prepared as outlined in Example 116. The startingmaterial was prepared as described in Example 1 using2-methoxyethanamine in place of methylamine in Step 5. MS m/z: 579.2(M+H⁺). ¹H NMR (400 MHz, CD₃OD): δ: 7.97 (1H, s), 6.91 (1H, s), 6.34(1H, m), 6.17 (1H, dd), 5.63 (1H, d), 4.59 (2H, m), 4.48 (1H, br s),4.37 (1H, br s), 4.29 (2H, m), 3.97 (6H, s), 3.48 (2H, s), 3.35 (3H, s),1.76 (6H, m).

Example 64: Synthesis of I-62

Compound I-62 was prepared as described in Example 17 using tert-butylpiperazine-1-carboxylate in place of N-ethylpiperizine in Step 2. MSm/z: 713.5 (M+H⁺).

Example 65: Synthesis of I-63 (Racemic)

Compound I-63 was prepared as described in Example 21. Intermediate 1was prepared as described in Example 2 using benzylamine in place of3-nitrobenzylamine in Step 2. MS m/z: 611.4 (M+H⁺).

Example 66: Synthesis of I-64

Compound I-64 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 usingthiazol-4-ylmethanamine in place of methylamine in Step 5. MS m/z: 544.4(M+H⁺).

Example 67: I-65

Step 1: Intermediate 2

A mixture of Intermediate 6 from Example 1 (100 mg, 0.25 mmol),2-nitrophenol (51.7 mg, 0.37 mmol), CS₂CO₃ (162 mg, 0.50 mmol) in NMP (5mL) was heated at 100° C. for 16 h. The mixture was cooled to ambienttemperature. Water was added and the resultant mixture was extractedwith EtOAc (35 mL×3). The combined organic layers were washed withwater, dried (Na₂SO₄), filtered and concentrated in vacuo. The crudeproduct was purified by column chromatography on silica gel (4%EtOAc/DCM) to afford 52.5 mg of the title compound.

Step 2: Intermediate 3

A mixture of Intermediate 2 (52.5 mg, 0.10 mmol), Fe (34.5 mg, 0.62mmol), NH₄Cl (33.3 mg, 0.62 mmol) in ethanol (4 mL) and H₂O (2 mL) washeated to reflux for 2 h. The mixture was allowed cool to ambienttemperature and filtered. The filtrate was concentrated in vacuo, waterwas added and the resultant mixture was extracted with EtOAc (35 mL×3).The combined organic layers were washed with saturated NaHCO₃, dried(Na₂SO₄), filtered and concentrated in vacuo. The crude product waspurified by column chromatography on silica gel (3% MeOH/DCM) to afford7.1 mg of the title compound. MS m/z: 476.3 (M+1)⁺

Step 3: I-65

To a cooled (0° C.) solution of Intermediate 3 (7.10 mg, 0.02 mmol) andDIPEA (3.9 mg, 0.03 mmol) in THF (4 mL) was added acryloyl chloride (1.6mg, 0.02 mmol). The reaction was stirred at 0° C. for 10 min after whichit was quenched with saturated NaHCO₃ and extracted with EtOAc (20mL×3). The combined organic layers were washed with saturated NaHCO₃,dried (Na₂SO₄), filtered and concentrated in vacuo. The residue waspurified by column chromatography on silica gel (4% EtOAc in DCM) toafford 3.0 mg of the title compound. MS m/z: 530.4 (M+1)⁺. NMR (400 MHz,CDCl₃): δ 3.45 (s, 3H), 3.94 (s, 6H), 4.57 (s, 2H), 6.08 (d, 1H), 6.41(dd, 1H), 6.61 (s, 1H), 6.67 (d, 1H), 7.05-7.11 (m, 1H), 7.20 (d, 1H),7.27-7.30 (m, 1H), 7.97 (s, 1H), 8.38 (d, 1H).

Example 68: I-66

The title compound (40 mg) was prepared as described in Example 84 usingtert-butyl (1R,2S)-2-aminocyclohexyl)carbamate in place of tert-butyl(1S,2R)-2-aminocyclohexyl)carbamate in Step 3. MS m/z: 611.1 (M+H⁺). ¹HNMR (400 MHz, DMSO-d6): δ: 7.96 (1H, d), 7.71 (1H, d), 7.28 (4H, m), 7.2(1H, m), 7.0 (1H, s), 6.3 (1H, dd), 6.05 (1H, dd), 5.55 (1H, dd), 5.12(1H, br s), 4.5 (2H, s), 3.96 (6H, s), 1.2-1.8 (8H, m).

Example 69: I-67

Step 1: Intermediate 2

A mixture of Intermediate 5 from Example 1 (150 mg, 0.45 mmol),benzene-1,2-diamine (48.5 mg, 0.45 mmol), CS₂CO₃ (292 mg, 0.90 mmol),Pd₂(dba)₃ (41.0 mg, 0.045 mmol) and xantphos (51.8 mg, 0.09 mmol) in1,4-dioxane (6 mL) was allowed to stir at 90° C. for 5 h under nitrogenatmosphere. The mixture was allowed to cool to ambient temperature.Water was added and the resultant mixture was extracted with EtOAc (30mL×3). The combined organic layers were washed with brine, dried(Na₂SO₄), filtered and concentrated in vacuo. The crude product waspurified by column chromatography on silica gel (3% MeOH/DCM) to afford88.5 mg of the title compound. MS m/z: 407.4 (M+1)⁺.

Step 2: I-67

To a cooled (0° C.) solution of Intermediate 2 (88.2 mg, 0.22 mmol) andEt₃N (87.8 mg, 0.87 mmol) in THF (6 mL) was added 2-chloroethanesulfonylchloride (35.4 mg, 0.22 mmol). The reaction was heated at 40° C. for 6h. Water was added and the resultant mixture was extracted with EtOAc(30 mL×3). The combined organic layers were dried (Na₂SO₄), filtered andconcentrated in vacuo. The crude product was purified by columnchromatography on silica gel (3% MeOH/DCM) to afford 8.8 mg of the titlecompound. MS m/z: 497.4 (M+1)⁺. ¹H NMR (400 MHz, CDCl3): δ 3.40 (s, 3H),3.79 (s, 6H), 4.64 (s, 2H), 5.86 (d, 1H), 6.14 (d, 1H), 6.39 (s, 1H),6.48 (d, 2H), 6.55 (dd, 9.6 Hz, 1H), 7.17-7.27 (m, 3H), 7.46-7.52 (m,2H), 7.95 (s, 1H), 8.12 (s, 1H).

Example 70: Synthesis of I-68 (Racemic)

Compound I-68 was prepared as described in Example 21 using Intermediate6 from Example 25 in place of Intermediate 2. MS m/z: 561.5 (M+H⁺).

Example 71: Synthesis of I-69 (Racemic)

Compound I-69 was prepared as described in Example 21 using Intermediate1 from Example 116 in place of Intermediate 2. MS m/z: 618.4 (M+H⁺).

Example 72: Synthesis of I-70 (Racemic)

Compound I-70 was prepared as described in Example 21. The startingmaterial was prepared as described in Example 2 using cyclobutylamine inplace of 3-nitrobenzylamine in Step 2. MS m/z: 575.3 (M+H⁺).

Example 73: Synthesis of I-71 (Racemic)

Compound I-71 was prepared as described in Example 10 using7-chloro-3-(3,5-dimethoxyphenyl)-1,4-dimethyl-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-onein place of Intermediate 8 in Step 1 (prepared as described in Example 2using 1-(2,4-dichloropyrimidin-5-yl)ethanone in place of Intermediate 2,methylamine in place of 3-nitrobenzylamine in Step 2, and omitting step6) and omitting steps 2-5. MS m/z: 481.5 (M+H⁺).

Example 74: Synthesis of I-72 (Racemic)

Compound I-72 was prepared as described in Example 4 using7-chloro-3-(3,5-dimethoxyphenyl)-1,4-dimethyl-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-onein place of Intermediate 1 in Step 1 (prepared as described in Example 2using 1-(2,4-dichloropyrimidin-5-yl)ethanone in place of Intermediate 2,methylamine in place of 3-nitrobenzylamine in Step 2, and omitting step6). MS m/z: 475.5 (M+H⁺).

Example 75: Synthesis of I-73 (Racemic)

Compound I-73 was prepared as described in Example 21. The startingmaterial was prepared as described in Example 2 using amonia in place of3-nitrobenzylamine in Step 2. MS m/z: 521.2 (M+H⁺).

Example 76: I-74

The title compound was prepared as described in Example 29 usingIntermediate 6 from Example 1 in place of Intermediate 5. MS m/z: 609.0(M+H⁺). ¹H NMR (400 MHz, CDCl₃): δ: 8.01 (1H, s), 7.95 (2H, m), 7.84(1H, s), 7.52 (1H, dd), 7.44 (1H, dd), 6.88 (1H, s), 6.47 (1H, dd), 6.38(1H, dd), 5.80 (1H, dd), 4.60 (2H, s), 3.95 (6H, s), 3.91 (3H, s), 3.35(3H, s).

Example 77: Synthesis of I-75

Compound I-75 was prepared as described in Example 102 using methylaminein place of cyclopropanamine in Step 5. MS m/z: 495.3 (M+H⁺).

Example 78: Synthesis of I-76 (Racemic)

Compound I-76 was prepared as described in Example 21. The startingmaterial was prepared as described in Example 2 using methylamine inplace of 3-nitrobenzylamine in Step 2, using2-chloro-3,5-dimethoxyaniline in place of 3,5-dimethoxyaniline in Step4, and skipping Step 6. MS m/z: 501.3 (M+H⁺).

Example 79; I-77

The title compound was prepared as described in Example 29 using5-bromo-2-nitroaniline in place of Intermediate 1 in Step 1. MS m/z:541.2 (M+H⁺). ¹H NMR (400 MHz, CDCl₃): δ: 8.00 (1H, s), 7.95 (1H, s),7.84 (1H, s), 7.77 (1H, s), 7.64 (1H, d), 7.53 (1H, m), 6.54 (2H, s),6.46 (1H, m), 6.41 (1H, m), 5.83 (1H, dd), 4.71 (2H, s), 3.93 (3H, s),3.78 (6H, s), 3.36 (3H, s).

Example 80: I-78

The title compound was prepared as outlined in Example 29 using2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiazole in place ofIntermediate 3 in Step 2. MS m/z: 544.2 (M+H⁺). ¹H NMR (400 MHz,DMSO-d6): δ: 9.94 (1H, s), 8.91 (1H, s), 8.52 (1H, s), 8.12 (1H, s),7.89 (1H, d), 7.74 (1H, d), 7.68 (1H, dd), 6.54 (3H, m), 6.41 (1H, m),6.27 (1H, dd), 5.79 (1H, dd), 4.69 (2H, s), 3.72 (6H, s), 3.26 (3H, s).

Example 81: Synthesis of I-79

Compound I-79 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 usingl-methylpiperidin-4-amine in place of methylamine in Step 5. MS m/z:468.3 (M+H⁺).

Example 82: Synthesis of I-80

Compound I-80 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 usingl-ethylpiperidin-4-amine in place of methylamine in Step 5. MS m/z:558.4 (M+H⁺).

Example 83: Synthesis of I-81

Compound I-81 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using tert-butyl4-aminopiperidine-1-carboxylate in place of methylamine in Step 5. Afinal BOC deprotection was performed (as described in Example 3, Step5). MS m/z: 530.3 (M+H⁺).

Example 84: Synthesis of I-82

Step 1: Intermediate 2

The title compound was prepared as described in Example 1 usingbenzylamine in place of methylamine in Step 5.

Step 2: Intermediate 3

The title compound was prepared as described in the literature(Chemistry & Biology 2010, 17, 285-295). Intermediate 2 (150 mg, 0.31mmol) and HOBT (78 mg, 0.51 mmoL) were dissolved in 4 mL of DMF andallowed to stir for 90 min. Ammonia (3.8 mL, 0.5 N in dioxane, 1.9 mmoL)was added and the reaction mixture was allowed to stir at ambienttemperature overnight. Solvent was removed under reduced pressure andthe reaction mixture partitioned with water, brine and chloroform. Theorganic phase was dried over sodium sulfate and the solvent removedunder reduced pressure to provide 300 mg of the title compound which wasused as is in the next reaction. MS m/z: 578.2 (M+H⁺).

Step 3: Intermediate 4

To a solution of Intermediate 3 (300 mg, 0.52 mmoL), tert-butyl((1S,2R)-2-aminocyclohexyl)carbamate (214 mg, 1 mmoL) and DIPEA (170 uL,1.56 mmoL) in 4 mL of DMF was heated to 70° C. for 4 h. The reactionmixture was allowed to cool and partitioned with water, brine and EtOAc.The organic phase was dried over sodium sulfate and the solvent underreduced pressure. The crude product was subjected to chromatography onsilica gel (eluting with a gradient of 0-75% EtOAc in heptane), whichgave 57 mg of the title compound. MS m/z: 657.2 (M+H⁺).

Step 4: Intermediate 5

To a solution of the Intermediate 4 (57 mg, 0.087 mmol) in 5 mL of DCMwas added 500 uL of HCl (4 N in dioxane) and the reaction was stirred atambient temperature for 1 h. Solvent was removed under reduced pressurewhich gave 77 mg of the title compound. MS m/z: 557.2 (M+H⁺).

Step 5: I-82

To a solution of Intermediate 5 (77 mg, 0.13 mmoL) and acrylic acid (9ul, 0.13 mmoL) in 500 uL of DMF was added DIPEA (114 uL, 0.65 mmoL) andHATU (49 mg, 0.13 mmoL). The reaction was allowed to stir at ambienttemperature for 15 mins then purified directly by flash chromatography(eluting with a gradient of 0-70% acetone in heptane), which gave 16 mgof the title compound. MS m/z: 611.1 (M+H⁺). ¹H NMR (400 MHz, DMSO-d6):δ: 7.96 (1H, d), 7.72 (1H, d), 7.28 (4H, m), 7.2 (1H, m), 7.0 (1H, s),6.31 (1H, dd), 6.05 (1H, dd), 5.55 (1H, dd), 5.12 (2H, br s), 4.5 (2H,s), 3.96 (6H, s), 1.2-1.8 (8H, m).

Example 85: Synthesis of I-83

Step 1: Intermediate 2

The title compound was prepared from Intermediate 1 according toliterature procedures (WO 01/29042; PCT/EP00/10088).

Step 2: Intermediate 3

Intermediate 2 (53 mg, 0.14 mmol) and phenylenediamine (74 mg, 0.69mmoL) were heated neat in a microwave at 120° C. for 30 min. The crudeproduct was subjected to flash chromatography on silica gel (elutingwith a gradient of 0-80% EtOAc in heptane) to provide 30 mg of the titlecompound. MS m/z: 415.1 (M+H⁺).

Step 3: I-83

To a cooled (−10° C.) solution of Intermediate 3 (30 mg, 0.072 mmoL) in500 uL of THF was added acryloyl chloride (6.1 μL, 0.076 mmoL). Thereaction was allowed to stir at -10° C. for 5 min, then treated withDIPEA (14 uL, 0.079 mmoL) and allowed to stir for an additional 5 min.The reaction mixture was partitioned between water, brine and theorganic layer separated and purified using flash chromatography onsilica gel (eluting with a gradient of 0-100% EtOAc in heptane), whichgave 25 mg of the title compound. MS m/z: 469.1 (M+H⁺). ¹H NMR (400 MHz,DMSO-d6): δ: 8.68 (1H, s), 8.12 (1H, s), 7.8 (1H, d), 7.64 (2H, d), 7.58(1H, d), 7.48 (1H, t), 7.2 (1H, t), 7.12 (1H, t), 6.5 (1H, dd), 6.3 (1H,dd), 5.79 (1H, dd), 4.57 (2H, s), 3.25 (3H, s).

Example 86: Synthesis of I-84

Compound I-84 was prepared as described in Example 31 using2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiazole in place ofIntermediate 3 in Step 2. MS m/z: 544.4 (M+H⁺).

Example 87: Synthesis of I-85

Compound I-85 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using tert-butyl4-aminopiperidine-1-carboxylate in place of methylamine in Step 5. Afinal BOC deprotection was performed (as described in Example 3, Step5). MS m/z: 598.2 (M+H⁺).

Example 88: Synthesis of I-86

Compound I-86 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 usingthiazol-4-ylmethanamine in place of methylamine in Step 5. MS m/z: 612.3(M+H⁺).

Example 89: Synthesis of I-87

Compound I-87 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using4-(aminomethyl)pyridin-2-amine in place of methylamine in Step 5. MSm/z: 553.4 (M+H⁺).

Example 90: Synthesis of I-88

Compound I-88 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using oxetan-3-amine inplace of methylamine in Step 5. MS m/z: 503.4 (M+H⁺).

Example 91: Synthesis of I-89 (Racemic)

Compound I-89 was prepared as described in Example 21.N-(3-((7-chloro-3-(2,6-dichloro-3,5-dimethoxyphenyl)-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)methyl)phenyl)methanesulfonamidewas used in place of Intermediate 2 and was prepared as described inExample 1 using N-(3-(aminomethyl)phenyl)methanesulfonamide in place ofmethylamine in Step 5. MS m/z: 704.3 (M+H⁺).

Example 92: Synthesis of I-90

Compound I-90 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using3-(aminomethyl)benzonitrile in place of methylamine in Step 5. MS m/z:562.5 (M+H⁺).

Example 93: I-91

The title compound was prepared as described in Example 5. The startingmaterial was prepared as described in Example 1 usingthiazol-2-ylmethanamine in place of methylamine in Step 5. MS m/z: 612.0(M+H⁺). ¹H NMR (400 MHz, CDCl₃): δ: 8.07 (1H, s), 7.11 (1H, d), 7.51(3H, m), 7.23 (2H, m), 6.90 (1H, s), 6.45 (1H, dd), 6.34 (1H, dd), 5.79(1H, dd), 5.50 (2H, s), 4.67 (2H, s), 3.96 (6H, s).

Example 94: I-92

The title compound was prepared as outlined in Example 116. The startingmaterial was prepared as described in Example 1 usingthiazol-2-ylmethanamine in place of methylamine in Step 5. MS m/z: 618.2(M+H⁺). ¹H NMR (400 MHz, CD₃OD): δ: 8.00 (1H, s), 7.70 (1H, d), 7.52(1H, d), 6.92 (1H, s), 6.32 (1H, dd), 6.16 (1H, d), 5.62 (1H, dd), 5.56(2H, br), 4.65 (2H, s), 3.97 (6H, s), 1.69 (6H, br), 1.51 (2H, br).

Example 95: Synthesis of I-93

Compound I-93 was prepared as described in Example 116 using((1R,2S)-2-aminocyclohexyl)carbamate in place of((1S,2R)-2-aminocyclohexyl)carbamate in Step 2. The starting materialwas prepared as described in Example 1 using thiazol-2-ylmethanamine inplace of methylamine in Step 5. MS m/z: 618.1 (M+H⁺).

Example 96: Synthesis of I-94

Compound I-94 was prepared as described in Example 116 using((1R,2S)-2-aminocyclohexyl)carbamate in place of((1S,2R)-2-aminocyclohexyl)carbamate in Step 2. The starting materialwas Intermediate 6 from Example 1. MS m/z: 535.1 (M+H⁺).

Example 97: Synthesis of I-95

Step 1: Intermediate 2

The title compound (645 mg, MS m/z: 570.0 (M+H⁺)) was prepared fromIntermediate 6 (Example 1), as described in Example 84 using 6-CF₃—HOBTin place of HOBT in Step 2.

Step 2: Intermediate 3

The title compound (440 mg, MS m/z: 581.2 (M+H⁺) was prepared fromIntermediate 2, as described in Example 84, Step 3.

Step 3: Intermediate 4

To a solution of the Intermediate 3 (440 mg, 0.76 mmoL) in 10 mL of DCMwas added 10 mL of HCl (4 N in dioxane) and the reaction was allowed tostir at ambient temperature for 2 h. Solvent was removed under reducedpressure and the resulting solid used directly in the subsequentreaction.

Step 4: I-95

The title compound (190 mg) was prepared as described in Example 84,Step 5. MS m/z: 535.1 (M+H)⁺. ¹H NMR (400 MHz, DMSO-d6): δ: 7.96 (1H,s), 7.77 (1H, d), 6.98 (1H, s), 6.7 (1H, br s), 6.32 (1H, br s), 6.04(1H, d), 5.55 (1H, dd), 4.44 (2H, s), 4.14 (2H, m), 3.97 (6H, s), 3.22(3H, s), 1.37-1.76 (8H, m).

Example 98: Synthesis of I-96

Compound I-96 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using tert-butyl2-aminoacetate in place of methylamine in Step 5. MS m/z: 561.5 (M+H⁺).

Example 99: Synthesis of I-97

Compound I-97 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using3-(aminomethyl)benzamide in place of methylamine in Step 5. MS m/z:580.4 (M+H⁺).

Example 100: Synthesis of I-98

Compound I-98 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 usingcyclopropylmethanamine in place of methylamine in Step 5. MS m/z: 501.5(M+H⁺).

Example 101: Synthesis of I-99

Compound I-99 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using2,2-difluoropropan-1-amine in place of methylamine in Step 5. MS m/z:511.6 (M+H⁺).

Example 102: I-100

Step 1: Intermediate 2

To a 125 mL sealed tube was added 3,5-dimethoxyaniline (2.00 g, 13.1mmol) into toluene (50 mL, 469 mmol). Acetic anhydride (1.36 mL, 14.4mmol) was added slowly and a precipitate formed. The reaction was heatedto 45° C. for 30 minutes and then the reaction was stirred overnight atroom temperature. The next day, the reaction was diluted with hexanesand filtered, rinsed with additional hexanes and dried under vacuum toafford 2.5 g of the title compound MS m/z: 196.2 (M+H)⁺.

Step 2: Intermediate 3

To a 250 mL round bottom flask was addedN-(3,5-dimethoxyphenyl)acetamide (3.20 g, 16.4 mmol) in DCM (75 mL). Thereaction mixture was cooled to 0° C. N-chlorosuccinimide (2.30 g, 17.2mmol) in 25 mL of DCM was slowly added and the mixture was allowed tostir at ambient temperature for 16 h. The reaction was concentrated andtaken up in a 1-1 mixture of hexanes-EtOAc. The resultant precipitatewas filtered the filtrate was concentrated, taken up in 1:1hexanes-EtOAc and the solid filtered to yield a second crop of solid.The precipitates collected were combined and purified on silica gel(eluting with 25% EtOAc in hexane to afford 1.50 g of the titlecompound. MS m/z: 230.2 (M+H)⁺.

Step 3: Intermediate 4

To a 125 mL sealed flask was added Intermediate 3 (1.90 g, 8.27 mmol) inEtOH (50 mL, 856 mmol) followed by potassium hydroxide (2.32 g, 41.4mmol) in 10 mL of water. The reaction was allowed to stir at 95° C. for16 h after which it was cooled and concentrated. The resultant oil waspartitioned between water and EtOAc, the organic layer was dried overMgSO4, filtered and concentrated to provide 1.00 g of the title compoundMS m/z: 188.1 (M+H)⁺.

Step 4: Synthesis of Intermediate 5

Into a sealed vessel was added Intermediate 4 (1.50 g, 8.00 mmol) in DMA(5 mL, 53.3 mmol), followed by DIPEA (820 μl, 8.80 mmol). The reactionmixture was allowed to stir for 10 min. at ambient temperature, and2,4-dichloro-5-(iodomethyl)pyrimidine from Example 1 (2.31 g, 8.00 mmol)was added. The reaction mixture was allowed to stir at 60° C. for 7.5 h,then 16 h at ambient temperature. The reaction mixture was concentratedand azeotroped several times with toluene. EtOAc was added and theorganic layer washed with brine (3×). The organic layers were dried withsodium sulfate, concentrated and purified by flash column chromatography(eluting with 5-25% EtOAc in heptane) to give 1.75 g of the titlecompound MS m/z: 348.2 (M+H)⁺.

Step 5: Synthesis of Intermediate 6

Intermediate 5 (1.50 g, 4.30 mmol) was dissolved in 1,4-dioxane (20 mL).cyclopropylmethanamine (612 mg, 8.61 mmol) and DIPEA (1.54 mL, 8.61mmol) were added. The solution was allowed to stir at 35° C. for 3 h.Water (20 mL) was added and the organic layer was separated, dried overmagnesium sulfate, filtered, and concentrated under reduced pressure.The residue was chromatographed through silica gel (eluted with DCM) toafford 1.17 g of the title compound MS m/z: 383.4 (M+H)⁺.

Step 6: Intermediate 7

Intermediate 6 (1.16 g, 3.03 mmol) was dissolved in DCM (15 mL).Triphosgene (873 mg, 3.33 mmol) was added in one portion. The yellowsolution turned partially cloudy then back to a solution. The mixturewas stirred at ambient temperature for 30 min. Triethylamine (2.11 mL,15.13 mmol) was added and the resulting suspension was stirred atambient temperature for 18 h. Formation of a chloroformate intermediatewas observed ([M+H]+=445 m/z). The suspension was transferred into apressure vessel and stirred at 50° C. for an additional 48 h. Themixture was allowed to cool and the organic phase was washed with water(20 mL) and saturated aqueous sodium bicarbonate (20 mL). The organiclayer was dried over magnesium sulfate, filtered, and concentrated underreduced pressure. The residue was chromatographed through silica gel(0-5% EtOAC in DCM) to afford 1.04 g of the title compound MS m/z: 409.4(M+H)⁺.

Step 7: Intermediate 8

Intermediate 7 was dissolved in 1,4-dioxane (3 mL), andbenzene-1,2-diamine (52.85 mg, 489 μmol) and TFA (244 μmol) were added.The solution was stirred at 95° C. for 20 h. The solution was dilutedwith EtOAc and washed with saturated aqueous sodium bicarbonate (5 mL)and brine (5 mL) then dried over magnesium sulfate, filtered, andconcentrated under reduced pressure. The residue was chromatographedthrough silica gel 5% MeOH in DCM to afford 100 mg of the title compoundMS m/z: 481.5 (M+H)⁺

Step 8: I-100

Intermediate 8 (98.0 mg, 204 μmol) was dissolved in DCM (2 mL). Et₃N(56.8 μl, 408 μmol) was added followed by acryloyl chloride (19.9 μl,245 μmol). The solution was stirred at ambient temperature for 30 minthen MeOH was added and the reaction mixture was concentrated underreduced pressure. The residue was chromatographed via reverse phasechromatography (pre-eluted with MeOH then 1 N NH₃ in methanol.Subsequent chromatography through silica gel (20-30% EtOAc in DCMprovided 21.0 mg of the title compound MS m/z: 535.6 (M+H)⁺. ¹H NMR (400MHz, DMSO-d6) δ 9.84 (s, 1H), 8.56 (s, 1H), 8.08 (d, 1H), 7.74 (dd, 1H),7.57 (d, 1H), 7.19 (td, 1H), 7.12 (td, 1H), 6.76 (dd, 2H), 6.52 (dd,1H), 6.28 (dd, 1H), 5.78 (dd, 1H), 4.78-4.67 (m, 1H), 4.47 (d, 1H),4.14-3.93 (m, 1H), 3.87 (s, 3H), 3.79 (m, 5H), 1.17 (m, 1H), 1.01-0.69(m, 1H), 0.48-0.10 (m, 4H).

Example 103: I-101

Step 1: Intermediate 2

Intermediate 7 from Example 102 (100 mg, 244 μmol) was combined with ciscyclohexane-1,2-diamine (69.75 mg, 610.85 μmol) and dissolved in1,4-dioxane (3 mL). The solution was stirred at 95° C. for 20 h. Thesolution was allowed cool to ambient temperature and was concentratedunder reduced pressure. The residue was chromatographed through silicagel (3% 1 N NH₃ in MeOH in DCM to afford 90 mg of the title compound. MSm/z: 487.5 (M+H)⁺.

Step 2: I-101

Intermediate 2 (90 mg, 185 μmol) was dissolved in DCM (2 mL).Triethylamine (51.5 μl, 370 μmol) was added, followed by acryloylchloride (18.0 μl, 222 μmol). The solution was allowed to stir atambient temperature for 30 min after which it was quenched with MeOH andconcentrated under reduced pressure. The residue was chromatographed viareverse phase HPLC and then on silica gel to afford the title compound.MS m/z: 541.6 (M+H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ 7.95 (s, 1H), 7.74 (d,1H), 6.78 (t, 1H), ), 6.73 (d, 1H), 6.60 (br s, 1H), 6.34 (dd, 2H), 6.04(dd, 1H), 5.56 (dd, 1H), 4.64 (d, 1H), 4.41 (d, 1H), 4.22 (br s, 1H),4.05 (br s, 1H), 3.90-3.80 (3, 7H), 1.84-1.03 (m, 10H), 0.92-0.73 (m,1H), 0.61-0.3 (m, 4H).

Example 104: Synthesis of I-102

Compound I-102 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using2-fluoropropan-1-amine in place of methylamine in Step 5. MS m/z: 493.6(M+H⁺).

Example 105: Synthesis of I-103

Compound I-103 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using2,2-difluoropropan-1-amine in place of methylamine in Step 5. MS m/z:579.5 (M+H⁺).

Example 106: Synthesis of I-104

Compound I-104 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using2,2-difluoropropan-1-amine in place of methylamine in Step 5 and2-chloro-3,5-dimethoxyaniline in place of 3,5-dimethoxyaniline in Step4. MS m/z: 545.5 (M+H⁺).

Example 107: Synthesis of I-105 (Racemic)

Compound I-105 was prepared as described in Example 21.7-chloro-3-(2,6-dichloro-3.5-dimethoxyphenyl)-1-(2,2-difluoroethyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-onewas used in place of Intermediate 2 and was prepared as described inExample 1 using 2,2-difluoropropan-1-amine in place of methylamine inStep 5. MS m/z: 585.5 (M+H⁺).

Example 108: Synthesis of I-106 (Racemic)

Compound I-106 was prepared as described in Example 21.7-chloro-3-(2,6-dichloro-3.5-dimethoxyphenyl)-1-(2-fluoroethyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-onewas used in place of Intermediate 2 and was prepared as described inExample 1 using 2-fluoropropan-1-amine in place of methylamine in Step5. MS m/z: 567.5 (M+H⁺).

Example 109: Synthesis of I-107 (Racemic)

Compound I-107 was prepared as described in Example 21.7-chloro-3-(2-chloro-3,5-dimethoxyphenyl)-1-(2,2-difluoroethyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-onewas used in place of Intermediate 2 and was prepared as described inExample 1 using 2,2-difluoropropan-1-amine in place of methylamine inStep 5 and 2-chloro-3,5-dimethoxyaniline in place of3,5-dimethoxyaniline in Step 4. MS m/z: 551.6 (M+H⁺).

Example 110: Synthesis of I-108

Step 1: Intermediate 1

To a 125 mL sealed tube was added 3,5-dimethoxyaniline (2.00 g, 13.1mmol) in toluene (50 mL). Acetic anhydride (1.36 mL, 14.4 mmol) wasadded and a precipitate began to form. The reaction was heated to 45° C.for 30 min and then cooled to ambient temperature and allowed to stir 16h. The reaction mixture was diluted with hexanes and filtered. The solidwas rinsed with additional hexanes and the filtrate dried under vacuumto afford 2.50 g of the title compound. MS m/z: 196.2 (M+H)⁺.

Step 2: Intermediate 2

To a 125 mL round bottom flask, selectfluor (5.99 g, 16.9 mmol) and MeCN(40 mL) were added and cooled to 0° C. Intermediate 1 (3.00 g, 15.4mmol) in 10 mL of MeCN was added at 0° C. and the reaction was allowedto stir and warm to ambient temperature for 16 h. The reaction wasconcentrated, EtOAc and H₂O were added, and the organic layer separated,dried over MgSO₄, filtered, and the filtrate concentrated. The resultantresidue was purified on silica gel (eluting with DCM-EtOAc). A secondpurification (eluting with 4% MeOH in DCM) provided 800 mg of the titlecompound. LC. MS m/z: 214.2 (M+H)⁺

Step 3: Intermediate 3

To a 125 mL round bottom was added Intermediate 2 (700 mg, 3.28 mmol) inEtOH (9.6 mL,) and potassium hydroxide (918 mg, 16.4 mmol) in 4 mL H₂Oand the reaction was sealed and heated for 20 h at 90° C. The reactionmixture was concentrated, H₂O and EtOAc were added, and the organiclayer separated, dried over MgSO4, filtered and concentrated. Theresultant solid was purified on silica gel (eluting with 40% EtOAc inhexane) to afford 450 mg of the title compound. LC. MS m/z: 172.1(M+H)⁺.

Step 4: Intermediate 4

To a sealed vessel was added Intermediate 3 (2.38 g, 13.9 mmol), DMA (20mL) and DIPEA (27.8 mmol, 2.59 mL). The reaction mixture was allowed tostir at ambient temperature for 15 min after which2,4-dichloro-5-(iodomethyl) pyrimidine (4.02 g, 13.9 mmol) was added.The reaction mixture was heated at 50° C. for 16 h after which it wasallowed cool to ambient temperature then partitioned between saturatedaqueous NH₄Cl (40 mL) and EtOAc (40 mL). The organic phase was collectedand the aqueous phase extracted one more time with EtOAc (40 mL). Theorganic phases were combined, dried over MgSO₄ and concentrated.Purification by flash chromatography on silica gel (eluting with 30%EtOAc in hexanes) provided 4.07 g of the title compound. MS m/z: 332.2(M+H)⁺.

Step 5: Intermediate 5

To a solution of Intermediate 4 (1.00 g, 3.00 mmol) in dioxane (10 mL),was added DIPEA (0.70 mL, 7.53 mmol) and 2 M methylamine in THF (4.52mL, 9.03 mmol). The reaction mixture was heated to 50° C. for 16 h.Additional methylamine was added (9.03 mmol, 4.52 mL) and the reactionmixture was allowed to stir at 50° C. for an additional 16 h. Thereaction mixture was concentrated and purified by flash chromatographyon silica gel (eluting with 50% EtOAC in heptanes) to provide 570 mg ofthe title compound. MS m/z: 327.3 (M+H)⁺.

Step 6: Intermediate 6

To a solution of Intermediate 5 (570 mg, 1.74 mmol) in THF (10 mL) atambient temperature was added triphosgene (1.92 mmol, 569 mg). Themixture was allowed to stir at ambient temperature for 30 minutes afterwhich Et₃N (0.74 mL, 5.23 mmol) was added and the reaction mixture wasallowed to stir at ambient temperature for 5 h. H₂O was added slowly tothe reaction mixture (5 mL) followed by saturated aqueous NaH₂CO₃ (20mL) until a pH of 10 was achieved.

The reaction mixture was extracted with EtOAc (2×20 mL), the combinedorganic phases were washed with brine and dried over MgSO4 and solventwas removed under reduced pressure. The resultant residue was tituratedwith Et₂O, and the solid filtered to provide 490 mg of the titlecompound. The filtrate was concentrated to provide an additional 100 mgof the title compound which was purified by flash chromatography onsilica gel (eluting with 10% EtOAc in DCM to provide an additional 20 mgof the title compound. MS m/z: 353.3 (M+H)⁺.

Step 7: Intermediate 7

To a solution of Intermediate 6 (250 mg, 0.71 mmol) in 1,4-dioxane (5mL) at ambient temperature was added 1,2-phenyl diamine (230 mg, 2.3mmol) followed 2 drops of TFA. The reaction mixture was heated 95° C.for 16 h after which it was allowed cool to ambient temperature andpartitioned between saturated aqueous NH₄Cl (10 mL) and EtOAc (20 mL).The organic phase was washed with brine, dried over MgSO₄, concentratedunder reduced pressure and the resultant residue purified by flashchromatography on silica gel (eluting with 20% EtOAc in DCM) to provide163 mg of title compound. MS m/z: 425.5 (M+H)⁺.

Step 8: I-108

To a solution of Intermediate 7 (163 mg, 0.38 mmol) in DCM (5 mL) wasadded Et₃N (0.05 mL, 0.38 mmol). The mixture was cooled to 0° C. andacryloyl chloride (34.8 mg, 0.38 mmol) was added dropwise. The reactionmixture was allowed to stir at 0° C. for 5 minutes then allowed warm toambient temperature for 30 min. The reaction mixture was partitionedbetween saturated aqueous NH₄Cl (10 mL) and EtOAc (20 mL). The organicphase was dried over MgSO4, concentrated, and the residue purified byflash chromatography on silica gel (eluting with 60% EtOAc in DMC) toprovide 32 mg of the title compound. MS m/z: 479.5 (M+H)⁺. ¹H NMR (400MHz, CDCl3): δ 3.4 (s, 3H), 3.80 (s, 3H), 3.90 (s, 3H), 4.6 (s, 2H),5.75 (d, 1H), 6.2 (m, 1H), 6.4 (m, 2H), 6.55 (d, 1H), 7.25 (m, 3H), 7.5(s, 1H), 7.8 (s, 1H), 7.9 (s, 1H), 8.4 (s, 1H).

Example 111: Synthesis of I-109

Compound I-109 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 usingcyclopropylmethanamine in place of methylamine in Step 5 and2-fluoro-3,5-dimethoxyaniline in place of 3,5-dimethoxyaniline in Step4. MS m/z: 519.6 (M+H⁺).

Example 112: Synthesis of I-110

Compound I-110 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using2-fluoropropan-1-amine in place of methylamine in Step 5 and2-chloro-3,5-dimethoxyaniline in place of 3,5-dimethoxyaniline in Step4. MS m/z: 527.5 (M+H⁺).

Example 113: Synthesis of I-111 (Racemic)

Compound I-111 was prepared as described in Example 21.7-chloro-3-(2-chloro-3,5-dimethoxyphenyl)-1-(2-fluoroethyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-onewas used in place of Intermediate 2 and was prepared as described inExample 1 using 2-fluoropropan-1-amine in place of methylamine in Step 5and 2-chloro-3,5-dimethoxyaniline in place of 3,5-dimethoxyaniline inStep 4. MS m/z: 533.5 (M+H⁺).

Example 114: Synthesis of I-112 (Racemic)

Compound I-112 was prepared as described in Example 21 usingcis-cyclopentane-1,2-diamine in place of cis-cyclohexane-1,2-diamine inStep 2. MS m/z: 521.3 (M+H⁺).

Example 115: Synthesis of I-113 (Racemic)

Compound I-113 was prepared as described in Example 21 usingcis-cyclohex-4-ene-1,2-diamine in place of cis-cyclohexane-1,2-diaminein Step 2. MS m/z: 533.4 (M+H⁺).

Example 116: Synthesis of I-114

Step 1: Intermediate 2

The title compound was prepared from Intermediate 1 according to amodified literature procedure (WO 2009; PCT/US2009/002401.) Intermediate1 was prepared as outlined in Example 1 using thiazol-4-ylmethanamine inplace of methylamine in Step 5. Intermediate 1 (540 mg, 1.11 mmoL), HOBT(340 mg, 2.23 mmoL) and NMP (735 uL, 6.68 mmoL) were charged in 12 mL ofdioxane; heated to 100° C. for 2 h. Solvent was removed under reducedpressure and water was added to cause precipitation, the resulting solidwas removed by filtration and the filtrate concentrated to provide 672mg of the title compound which was used directly in the next reaction.MS m/z: 585.0 (M+H⁺).

Step 2: Intermediate 3

A solution of Intermediate 2 (672 mg, 1.15 mmoL), tert-butyl((1S,2R)-2-aminocyclohexyl)carbamate (492 mg, 2.3 mmoL), and NMP (400uL, 3.67 mmoL) in 12 mL of DMF/1.2 mL of NMP was heated to 100° C. for16 h. Solvent was removed under reduced pressure and the crude productwas subjected to chromatography on silica gel (eluting with a gradientof 0-100% EtOAc in heptane), which gave 650 mg of the title compound. MSm/z: 664.1 (M+H⁺).

Step 3: Intermediate 4

To a solution of the Intermediate 3 (650 mg, 0.98 mmoL) in 10 mL of DCMwas added 10 mL of HCl (4 N in dioxane) and the reaction was stirred atambient temperature for 2 h. The solvent was removed under reducedpressure to provide the title compound. MS m/z: 564.0 (M+H⁺).

Step 4: I-114

A solution of Intermediate 4 (552 mg, 0.98 mmoL) in 3 mL of DMF wascooled in ice-water/methanol bath and acrylic acid (62 ul, 0.98 mmoL)was added. To the mixture was added DIPEA (1 mL, 5.9 mmoL) and then HATU(345 mg, 0.98 mmoL). The reaction was allowed to stir at ambienttemperature for 15 min and purified directly by flash chromatography(eluting with a gradient of 0-100% acetone in heptane), which gave 515mg of the title compound. MS m/z: 618.0 (M+H⁺).

Example 117: Synthesis of I-115

Step 1: Intermediate 1

To a 1000 mL round bottom flask was added selectfluor (13.5 g, 38.2mmol) and MeCN (400 mL). The suspension was cooled to 0° C. and methyl3,5-dimethoxybenzoate (5.00 g, 25.5 mmol) in minimal MeCN was addedslowly over 10 minutes. The reaction mixture was allowed to stir andwarm to room temperature over 2 days after which a saturated aqueoussolution of sodium carbonate was added and the reaction mixture wasallowed to stir for 15 minutes and MeCN was removed under reducedpressure. Water and EtOAc were added and the organic layer was washedwith an aqueous saturated solution of NaCl (3×). The organic layer wasdried over MgSO₄, filtered, and dried concentrated and purified throughflash chromatography on silica gel: (eluting with 30 to 50% hexanes inDCM) to afford 1.0 g of the title compound MS m/z: 233.3 (M+H⁺).

Step 2: Synthesis Intermediate 2

To a 125 mL sealed tube was added methyl Intermediate 1 (1.00 g, 4.31mmol) and KOH (507 mg, 9.04 mmol) in EtOH (25 mL). The reaction mixturewas heated to 90° C. for 20 h and then cooled and concentrated. Waterwas added and the resultant solution was treated with 1 N HCl until apH<3 was achieved. A white precipitate formed which was filtered andrinsed with water. The solid was dissolved in EtOAc, dried over MgSO4,and the organic filtrate was concentrated to afford 600 mg of the titlecompound MS m/z: 219.2 (M+H⁺).

Step 3: Intermediate 3

To a 125 mL round bottom tube was added Intermediate 2 (0.62 g, 2.84mmol), diphenyl phosphorazidate (647 μl, 2.98 mmol), Et₃N (416 μl, 2.98mmol) and 2-methylpropan-2-ol (299 μl, 3.13 mmol) in toluene (5 mL) andthe reaction mixture was sealed and heated to 70° C. for 2 h. Thereaction mixture was allowed cool and toluene was removed under reducedpressure. EtOAc was added and the organic phase washed with sequentiallywith saturated aqueous Na₂CO₃ (2×) and saturated aqueous NaCl (2×). Theorganic phase was dried over MgSO₄, concentrated, and the resultantresidue was purified on silica gel (eluting with 100% DCM) to give 500mg of the title compound. ¹H NMR (500 MHz, DMSO-d6): δ 8.81 (s, 1H),6.89 (t, 1H), 3.32 (s, 6H), 1.42 (s, 9H).

Step 4: Intermediate 4

To a 125 mL round bottom flask was added Intermediate 3 (500 mg, 1.73mmol), 4 N HCl in dioxane (8.64 mL, 34.6 mmol) and the reaction mixturewas allowed to stir at ambient temperature for 5 h after which it wasconcentrated to give the HCl salt of title compound. ¹H NMR (500 MHz,DMSO-d6): δ 6.17 (s, 2H), 3.77 (s, 6H).

Step 5: Intermediate 5

To a 125 mL tube was added Intermediate 4 (360 mg, 1.60 mmol)2,4-dichloro-5-(iodomethyl)pyrimidine (461 mg, 1.60 mmol) and DIPEA (848μl, 4.79 mmol) in DMA (4 mL). The tube was sealed and the reactionmixture heated to 65° C. with stirring for 4 h after which it wascooled, concentrated and co-evaporated with toluene several times. Theresultant oil was dissolved in EtOAc, and solids were removed byfiltration. The filtrate was concentrated, dissolved in DCM and purifiedthrough flash chromatography on silica gel (eluting with EtOAc/hexanes)to provide 350 mg of the title compound. MS m/z: 350.3 (M+H⁺).

Step 6: Intermediate 6

Intermediate 5 (128 mg, 365.56 μmol) and 2,2-difluoroethanamine (59.3mg, 731 μmol) were dissolved in 1,4-dioxane. DIPEA (131 μl, 731 μmol)was added and the solution was allowed to stir at 45° C. for 5 h then at40° C. for 2 d. The solution was concentrated under reduced pressure andthe resultant residue was purified through flash chromatography onsilica gel (30% EtOAc in hexanes) to afford 105 mg of the titlecompound. MS m/z: 395.4 (M+H⁺).

Step 7: Intermediate 7

Intermediate 6 (103 mg, 261 μmol) was dissolved in DCM (2 mL) andtriphosgene (85.2 mg, 287 μmol) was added. The solution was allowed tostir at ambient temperature for 1 h. Et₃N (182 μl, 1.30 mmol) was addedand the solution was allowed to stir an additional 1 h. LCMS showeddisappearance of Intermediate 6 and formation of the carbamic chlorideintermediate. The reaction mixture was heated at 50° C. for 72 h afterwhich it was allowed cool to ambient temperature and water (2 mL) wasadded followed by saturated aqueous sodium bicarbonate (3 mL). Themixture was allowed to stir at ambient temperature for 30 min then wasextracted with DCM and the organic layers dried over MgSO₄, filtered,and concentrated under reduced pressure. The resultant residue waspurified through flash chromatography on silica gel (eluting with DCM)to afford 80 mg of the title compound. MS m/z: 421.4 (M+H⁺).

Step 8: Intermediate 8

Intermediate 7 (32.0 mg, 76.1 μmol) was dissolved in 1,4-dioxane (0.5mL) and cis-cyclohexane-1,2-diamine (21.7 mg, 190 μmol) was added. Thesolution was allowed to stir in a sealed tube at 90° C. for 18 h afterwhich it was concentrated under reduced pressure and the resultantresidue was purified through flash chromatography on silica gel (elutingwith 10% MeOH in DCM) to afford 33 mg of the title compound. MS m/z:499.6 (M+H⁺).

Step 9: I-115

Intermediate 8 (30 mg, 60.2 μmol) was dissolved in DCM. The solution wascooled to 0° C. and Et₃N (16.8 μl, 120 μmol) was added, followed byacryloyl chloride (4.87 μl, 60.2 μmol). The suspension was stirred at 0°C. for 45 min. Methanol was added and the resulting solution wasconcentrated under reduced pressure. The residue was chromatographedthrough silica gel (eluting with 60% EtOAc in DCM) to afford 20 mg ofthe title compound. MS m/z: 553.6 (M+H⁺). ¹H NMR (400 MHz, DMSO-d6) δ8.01 (s, 1H), 7.76 (m, 1H), 7.07 (t, 1H), 7.00-6.60 (m, 1H), 6.48-6.11(m, 2H), 6.03 (dd, 1H), 5.54 (dd, 1H), 4.57 (s, 2H), 4.37 (m, 2H), 4.18(m, 2H), 4.09 (s, 6H), 1.86-1.24 (m, 8H).

Example 118: Synthesis of I-116

Step 1: Intermediate 2

Intermediate 7 from Example 117 was dissolved in 1,4-dioxane (0.5 mL)and benzene-1,2-diamine (16.5 mg, 152 μmol) and 2 drops of TFA wereadded. The vessel was sealed and the solution was stirred at 90° C. for18 h after which the reaction mixture was concentrated under reducedpressure and the resultant residue was purified through chromatographyon silica gel (60% EtOAc in DCM) to afford 21.0 mg of the titlecompound. MS m/z: 493.5 (M+H⁺).

Step 2: I-116

Intermediate 2 (20.0 mg, 40.6 μmol) was dissolved in DCM and thesolution cooled to 0° C. Et₃N (11.3 μl, 81.2 μmol) was added followed byacryloyl chloride (3.21 μl, 40.6 μmol). The suspension was allowed tostir at 0° C. for 1.5 h after which methanol was added and the mixturewas concentrated under reduced pressure. The resultant residue waschromatographed through silica gel (30% EtOAc in DCM/to afford 11.0 mgof the title compound MS m/z: 547.6 (M+H⁺). 1H NMR (400 MHz, DMSO-d6) δ9.82 (s, 1H), 8.75 (s, 1H), 8.14 (s, 1H), 7.72 (dd, 1H), 7.65-7.53 (m,1H), 7.25-7.01 (m, 3H), 6.51 (dd, 1H), 6.40-6.04 (m, 2H), 5.78 (dd, 1H),4.65 (s, 2H), 4.32 (m, 2H), 3.90 (s, 6H).

Example 119; Synthesis of I-117

Step 1: Intermediate 2

To a 10 mL tube was added Intermediate 6 from Example 1 (140 mg, 347μmol), potassium carbonate (240 mg, 1.73 mmol), Brett Phos (16.2 mg,17.3 μmol), 1-methyl-1H-pyrazole-3,4-diamine dihydrochloride (64.2 mg,347 μmol) in tert-butanol (6 ml, 62.7 mmol) and the reaction mixture wassealed and purged with nitrogen. The mixture was heated to 110° C. for 8h after which it was allowed to cool to ambient temperature and waterwas added. The resultant precipitate was collected via filtration. Andthe filtrate was extracted with DCM. The organic layer was dried overMgSO4, filtered, and concentrated. The combined solids were purifiedthrough flash chromatography on silica gel (MeOH/DCM) to provide 48.0 mgof the title compound. MS m/z: 479.4 (M+H)⁺. ¹H NMR was consistent witha single isomer although 2D NMR correlations did not confirm whichisomer formed. Material was assigned regiochemical assignment ofIntermediate 2 based on presumed decreased electrophilicity of theN-methylpyrazole amine and material was carried on to the next step.

Step 2: I-117

Intermediate 2 (48.0 mg, 100 μmol) was dissolved in DCM (1 ml). Thesuspension was cooled to 0° C. Et₃N (27.9 μl, 200 μmol) was addedfollowed by acryloyl chloride (7.92 μl, 100 μmol). The resultantsuspension was allowed to stir at 0° C. for 90 min. Methanol was addedand the mixture was concentrated under reduced pressure. The resultantresidue was purified through flash chromatography on silica gel (30% DCMin EtOAc) to afford 30.0 mg of the tittle compound. MS m/z: 533.5(M+H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ 10.74 (s, 1H), 8.95 (s, 1H), 8.10(d, 2H), 7.01 (s, 1H), 6.53 (m, 1H), 6.36 (m, 1H), 5.82 (dd, 1H), 4.53(s, 2H), 3.98 (s, 6H), 3.81 (s, 3H).

Example 120: I-119

The title compound was prepared as outlined in the Example 116 usingtert-butyl ((1R,2S)-2-aminocyclohexyl)carbamate in place of tert-butyl((1S,2R)-2-aminocyclohexyl)carbamate in Step 2. MS m/z: 618.0 (M+H⁺). ¹HNMR (400 MHz, DMSO-d6): δ: 9.01 (1H, d), 8.0 (1H, s), 7.69 (1H, d), 6.99(1H, s), 6.3 (1H, dd), 6.05 (1H, dd), 5.55 (1H, dd), 5.26 (2H, bs), 4.52(2H, s), 3.96 (6H, s), 3.3 (4H, m), 2.16 (4H, m), 1.9 (4H, m).

Example 121: Synthesis of I-120

Compound I-120 was prepared as described in Example 53 using ethylaminein place of cyclopropanamine in Step 1 and in place of methylamine inStep 2. MS m/z: 367.3 (M+H⁺).

Example 122: Synthesis of I-121

Compound I-121 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using(1H-pyrazol-4-yl)methanamine in place of methylamine in Step 5. MS m/z:527.5 (M+H⁺).

Example 123; Synthesis of I-122

Step 1, Intermediate 2

Intermediate 1 (57.0 mg, 0.034 mmol) and PMB-NH₂ (0.13 mL, 1.01 mmol) inDMF (2.0 mL) were heated at 110° C. for 4 h. DMF was removed in vacuo,and the resultant residue was dissolved in DCM (5 mL), followed withaddition of AcOH (0.1 mL) and N-ethylpiperazine (0.10 mL, 7.90 mmol).After 1 h, NaHB(OAc)₃ (100 mg, 0.47 mmol) was added and the reaction wasallowed to stir for 16 h. EtOAc was added and the organic phase washedwith saturated aqueous NaHCO₃ and brine, dried (Na₂SO₄), filtered andconcentrated in vacuo. Flash chromatography on silica gel, provided 80mg of the title compound. MS m/z: 385.2 (M+H⁺).

Step 2: Intermediate 3

Intermediate 2 (80.0 mg, 0.21 mmol) was treated with 20% (v/v) TFA inDCM at ambient temperature and the reaction mixture was allowed to stirfor 30 min. The reaction mixture was concentrated in vacuo and theresultant residue was treated with silica supported carbonate, filtered,and concentrated to afford quantitative yield of the title compoundwhich was used without further purification. MS m/z: 265.2 (M+H⁺).

Step 3, Intermediate 4

To a solution of Intermediate 3 (56.0 mg, 0.21 mmol) in DMF (4.0 mL) wasadded (BOC)₂O (100 mg, 0.46 mmol) and DMAP (10.0 mg, 0.08 mmol). Thereaction was stirred at ambient temperature overnight. DMF was removedunder reduced pressure and the product was isolated by silica gelchromatography (98.0 mg). MS m/z: 465.3 (M+H⁺).

Step 4, Intermediate 5

To a solution of Intermediate 4 (98.0 mg, 0.21 mmol) in MeOH (5 mL) at0° C. was added NiCl₂-6H₂O and NaBH₄. After 30 min, the reaction wasquenched through addition of water, filtered through celite, andconcentrated to afford 62.0 mg of the title compound which was usedwithout further purification. MS m/z: 335.3 (M+H⁺).

Step 5: I-122

The title compound was prepared as described in Example 5 usingIntermediate 5 from Example 1. MS m/z: 587.3 (M+H⁺). ¹H NMR (400 MHz,CD₃OD): δ: 8.00 (1H, s), 7.79 (1H, d), 7.64 (1H, s), 7.55 (1H, d), 7.29(1H, dd), 6.53 (2H, s), 6.45 (2H, m), 5.81 (1H, dd), 4.71 (2H, s), 3.78(6H, s), 3.77 (2H, s), 3.35 (3H, s), 3.35 (4H, d) 3.30 (4H, d) 3.19 (2H,q), 1.32 (3H, t).

Example 124: Synthesis of I-123

Compound I-123 was prepared as described in Example 128 using succinicacid in place of N-biotinyl-NH—(PEG)₂-COOH in Step 2. MS m/z: 713.5(M+H⁺).

Example 125: Synthesis of I-124

Compound I-124 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using tert-butyl4-(aminomethyl)-1H-pyrazole-1-carboxylate in place of methylamine inStep 5. A final BOC deprotection step was performed (as described inExample 3, Step 5). MS m/z: 595.4 (M+H⁺).

Example 126: Synthesis of I-125

Compound I-125 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using amonia in place ofmethylamine in Step 5. MS m/z: 447.4 (M+H⁺).

Example 127: Synthesis of I-126

Step 1: Intermediate 2

To a solution of Intermediate 1 (2.00 g, 28.53 mmol) in 50 mL ofacetone/H₂O (4:1) was added NMO (10 g, 50% aqueous solution, 42.7 mmol)and K₂OsO₄.2H₂O (210 mg, 0.57 mmol). The reaction mixture was allowed tostir at ambient temperature for 16 h. Na₂SO₃ (8.0 g) was added and theresultant mixture was allowed to stir for 10 min. The reaction mixturewas evaporated to dryness and the residue taken up in EtOAc. Theresultant suspension was filtered and the filtrate was evaporated todryness to afford 2.50 g of the title compound.

Step 2: Intermediate 3

To an ice cooled solution of Intermediate 2 (2.50 g, 24.0 mmol) and Et₃N(12.2 g, 121 mmol) in DCM (50 mL) was added methanesulfonyl chloride(13.8 g, 121 mmol). The mixture was allowed stirred at 0° C. for 30 min.Saturated aqueous Na₂CO₃ solution was added and the reaction mixture waspartitioned between DCM and water. The organic layer was separated,washed with brine, dried over anhydrous Na₂SO₄, and evaporated. Thecrude solid was re-crystallized with ethanol to afford 4.83 g of thetitle compound. ¹H NMR (400 MHz, CDCl₃): δ 3.15 (s, 6H), 3.98-4.02 (m,2H), 4.14-4.19 (m, 2H), 5.18-5.21 (m, 2H).

Step 3: Intermediate 4

To a solution of Intermediate 3 (4.83 g, 18.6 mmol) in DMF (50 mL) wasadded 15-crown-5 (0.40 g, 1.82 mmol) and NaN₃ (6.03 g, 92.8 mmol). Themixture was heated to 100° C. under N₂ overnight. The reaction solutionwas diluted with EtOAc, washed with water and brine, dried overanhydrous Na₂SO₄ and evaporated to dryness to afford the 2.60 g of thetitle compound.

Step 4: Intermediate 5

To a solution of Intermediate 4 (2.60 g, 16.9 mmol) in MeOH (15 mL) wasadded Pd/C (10%, w/w, 0.78 g) and the reaction mixture was allowed tostir at ambient temperature for 16 h. The reaction mixture was filteredand the filtrate was concentrated to afford 1.45 g of the title compoundwhich was used without further purification.

Step 5: Intermediate 6

To a solution of Intermediate 5 (0.90 g, 8.81 mmol) and Intermediate 6from Example 1 (1.42 g, 0.40 eq) in NMP (10 mL) was added DIPEA (2.30 g,17.8 mmol). The reaction mixture was heated at 100° C. for 16 h undernitrogen. The reaction mixture was cooled to ambient temperature, waterwas added and the aqueous layer was extracted with EtOAc (40 mL×3). Thecombined organic layers were dried (anhydrous Na₂SO₄), filtered andconcentrated. Purification by column chromatography on silica gel (5%MeOH in DCM) afforded 162 mg of the title compound. MS m/z: 469.4(M+H)⁺.

Step 9: I-126

To an ice-cooled solution of Intermediate 6 (162 mg, 0.35 mmol) and Et₃N(37.5 mg, 0.41 mmol) in DCM (10 mL) was added acryloyl chloride (41.9mg, 0.41 mmol). The mixture was stirred at 0° C. for 10 min. Thereaction mixture was quenched with saturated aqueous NaHCO₃ and theresultant mixture was extracted with EtOAc. The organic phase was dried,concentrated, and the residue was purified by column chromatography onsilica gel (3% MeOH in DCM) to afford 112 mg of the title compound. MSm/z: 523.4 (M+H)⁺. ¹H NMR (400 MHz, CDCl₃) δ: 3.39 (s, 3H), 3.71-3.81(m, 2H), 3.94 (s, 6H), 4.17-4.21 (m, 2H), 4.52 (s, 2H), 4.73-4.79 (m,2H), 5.62-5.65 (m, 2H), 6.02-6.07 (m, 1H), 6.26 (d, 1H), 6.32 (br, 1H),6.60 (s, 1H), 7.84 (s, 1H).

Example 128: Synthesis of I-127

To a solution of I-62 (30.0 mg, 0.042 mmol) in 2 mL of DCM was added 200uL of TFA and the reaction mixture was allowed to stir for 1 h atambient temperature. Solvent was removed under reduced pressure and theresulting solid was dissolved in 300 ul of DMF. To this solution wasadded the N-biotinyl-NH—(PEG)₂-COOH.DIPEA (35 mg, 0.049 mmol), HOBT (7mg, 0.046 mmol), EDC (9 mg, 0.046 mmol) and NMM (30 uL, 0.28 mmol), andthe mixture was allowed to stir at ambient temperature for 16 h. Thecrude product was purified by reverse phase preparative HPLC (elutingwith a gradient of 10-90% MeCN in H₂O with 0.1% aqueous TFA), which gave22 mg of the title compound. MS m/z: 1155.3 (M+H⁺). ¹H NMR (400 MHz,DMSO-d6): δ: 9.73 (1H, s), 8.05 (1H, s), 7.77 (2H, m), 7.47 (1H, d), 7.3(1H, s), 7.0 (1H, s), 6.86 (1H, dd), 6.5 (1H, dd), 6.45 (1H, br s), 6.25(1H, dd), 5.77 (1H, dd), 4.51 (2H, s), 4.3 (1H, m), 4.1-3.0 (31H, m),2.8 (1H, dd), 2.34 (3H, m), 2.08 (5H, tt), 1.73 (3H, m), 1.6 (6H, m),1.5-1.2 (8H, m).

Example 129: Synthesis of I-128

Step 1: Intermediate 2

The title compound was prepared from Intermediate 1 according toliterature procedures (WO 01/19825; PCT/US00/17037; WO 2008/051820;PCT/US2007/081899).

Step 2: Intermediate 3

A solution of Intermediate 2 (200 mg, 1.04 mmol) and mono-BOC phenylenediamine (450 mg, 2.07 mmol) in 9 mL of 1:2 THF/IPA was heated to 100° C.for 1 h. The solvent was removed under reduced pressure and the reactionmixture was partitioned between water and EtOAc. The organic layer wasdried with Na₂SO₄, filtered and concentrated under reduced pressure. Thecrude product was subjected to chromatography on silica gel (elutingwith a gradient of 0-50% EtOAc in heptane), which gave 150 mg of thetitle compound. MS m/z: 361.1 (M+H⁺)

Step 3: Intermediate 4

To a solution of Intermediate 3 (250 mg, 0.69 mmol) in 6 mL of dioxanewas added a solution of sodium hydrosulfite (1.5 g, 8.68 mmol in 12 mLof water) and 500 ul of concentrated ammonium hydroxide. A solid formedwhich was sonicated until a solution was achieved. The solution wasconcentrated and partitioned with brine and EtOAc; the organic layer wasdried with Na₂SO₄, filtered and the solvent was removed under reducedpressure to provide 260 mg of the title compound. MS m/z: 331.1 (M+H⁺)

Step 4: Intermediate 5

A solution of Intermediate 4 (260 mg, 0.79 mmol) and ethyl2-(3,5-dimethoxyphenyl)-2-oxoacetate (220 mg, 0.94 mmol) in 10 mL ofEtOH containing 400 ul of acetic acid; was refluxed for 16 h. A solidformed which was filtered to provide 150 mg of the title compound. MSm/z: 505.2 (M+H⁺)

Step 6: I-128

To a solution of intermediate 5 (150 mg, 0.3 mmol) in 10 mL of DCM wasadded 2 mL of TFA and the reaction mixture was allowed to stir for 2 hat ambient temperature. Solvent was removed under reduced pressure andpartitioned with cold (0° C.) saturated NaHCO₃ and EtOAc. The solidformed was filtered to provide 122 mg. of free amine intermediate. MSm/z: 405.2 (M+H⁺). The solid was then suspended in 5 mL of THF and 500uL of DMF and the solution was cooled in an ice-water/methanol bath.Acryloyl chloride (20 μL, 0.23 mmol) was added and the reaction mixturewas allowed to stir at -10° C. for 5 min after which it was treated withDIPEA (50 uL, 0.276 mmol) and allowed to stir at ambient temperature for30 min. The solvent was reduced in volume and the crude product wassubjected to chromatography on silica gel (eluting with a gradient of0-75% EtOAc in heptane). The title compound was purified further byreverse phase preparative HPLC (eluting with a gradient of 10-90%acetonitrile in H₂O with 0.1% aqueous TFA), which gave 10 mg of thetitle compound. MS m/z: 459.0 (M+H⁺). ¹H NMR (400 MHz, DMSO-d6): δ: 9.87(1H, s), 9.43 (1H, s), 8.87 (1H, s), 7.81 (1H, d), 7.649 (1H, d), 7.408(2H, d), 7.25 (2H, m), 6.64 (1H, dd), 6.5 (1H, dd), 6.3 (1H, dd), 5.78(1H, dd), 3.8 (6H, s), 3.50 (3H, s).

Example 130: Synthesis of I-129 (Racemic)

Compound I-129 was prepared as described in Example 21.7-chloro-1-(cyclopropylmethyl)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-onewas used in place of Intermediate 2 and was prepared as described inExample 1 using cyclopropylmethanamine in place of methylamine in Step5. MS m/z: 575.5 (M+H⁺).

Example 131: Synthesis of I-130

Compound I-130 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 usingcyclopropylmethanamine in place of methylamine in Step 5. MS m/z: 569.5(M+H⁺).

Example 132: Synthesis of I-131

Compound I-131 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using2,6-difluoro-3,5-dimethoxyaniline in place of 3,5-dimethoxyaniline inStep 4. MS m/z: 497.5 (M+H⁺).

Example 133: Synthesis of I-132

Step 1: Intermediate 3

Intermediate 4 from Example 1 (380 mg, 1.21 mmol), Intermediate 2(prepared as described in U.S. Pat. No. 7,713,994) (251 mg, 1.81 mmol)and DIPEA (642 μl, 3.63 mmol) were combined in 1,4-dioxane (5 mL). Thereaction vessel was sealed and heated to 50° C. for 16 h after which itwas concentrated and the resultant residue taken up in DCM. Solids werefiltered and the filtrate purified through flash chromatography onsilica gel (eluting with 1-5% MeOH in EtOAc) to provide 160 mg of thetitle compound with 20% regioisomer impurity which was carried onwithout further purification. MS m/z: 416.4 (M+H⁺).

Step 4: Intermediate 4

Intermediate 3 (160 mg, 385 μmol) and triphosgene (120 mg, 404 μmol)were combined in THF (5 mL) and allowed to stir for 30 min. Et₃N (154μl, 1.15 mmol) was added and the reaction was heated to 50° C. for 16 hafter which it was allowed cool to ambient temperature and the resultantresidue was purified through flash chromatography on silica gel (elutingwith MeOH/DCM) to afford 60 mg of the title compound MS m/z: 442.4(M+H⁺).

Step 5: Intermediate 5

Intermediate 4 (56 mg, 127 μmol) was dissolved in MeCN (0.3 mL) and DCM(0.3 mL). The solution was cooled to 0° C. and sulfuryl dichloride (20.6μl, 254 μmol) was added. The mixture was allowed to stir at 0° C. for 2h. Water was added followed by saturated aqueous sodium bicarbonate andthe mixture was allowed to stir for an additional 20 min at ambienttemperature. The reaction mixture was extracted with DCM and thecombined organic layers were dried over MgSO₄, and concentrated underreduced pressure and purified by flash chromatograph on silica gel(eluting with 30% EtOAc in DCM) to provide 16 mg of the title compoundMS m/z: 510 (M+H⁺).

Step 6: Intermediate 6

Intermediate 5 (16.0 mg, 31.3 μmol) was suspended in 1,4-dioxane (0.5mL) and (cis)-cyclohexane-1,2-diamine (7.15 mg, 62.3 μmol) and Et₃N(13.1 μl, 94.0 μmol) were added. The resultant suspension was allowed tostir at 95° C. for 6 h after which the reaction mixture was concentratedunder reduced pressure and the resultant residue was purified by flashchromatography on silica gel (eluting with 15% MeOH in DCM) to afford15.0 mg of the title compound.

Intermediate 6 (15.0 mg, 25.5 μmol) was suspended in DCM (0.25 mL) andEt₃N (7.11 μl, 51.0 μmol) was added. The mixture was cooled to 0° C. andacryloyl chloride (1.60 μl, 25.5 μmol) was added. The mixture wasallowed to stir at 0° C. for 30 min then MeOH (3 mL) was added. Thesolution was concentrated under reduced pressure and the resultantresidue was purified through flash chromatography on silica gel (10%MeOH in DCM) to provide 5 mg of the title compound. MS m/z: 642.6(M+H⁺). ¹H NMR (400 MHz, DMSO-d6) δ 8.01 (m, 1H), 7.68 (d, 1H), 7.61 (d,1H), 7.00 (s, 1H), 6.74 (m, 1H), 6.40-6.23 (m, 1H), 6.17-5.98 (m, 3H),5.54 (dt, 1H), 4.91 (s, 2H), 4.53 (s, 2H), 3.96 (s, 5H), 4.01 (m, 1H),3.36 (s, 6H), 1.87-1.11 (m, 8H).

Example 134: Synthesis of I-133

Compound I-133 was prepared as described in Example 21.1-((2-aminopyridin-4-yl)methyl)-7-chloro-3-(2,6-dichloro-3,5-dimethoxyphenyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-onewas used in place of Intermediate 2 and was prepared as described inExample 1 using 4-(aminomethyl)pyridin-2-amine in place of methylaminein Step 5. MS m/z: 627.5 (M+H⁺).

Example 135: Synthesis of I-134

Compound I-134 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using(2-methylthiazol-4-yl)methanamine in place of methylamine in Step 5. MSm/z: 626.4 (M+H⁺).

Example 136: Synthesis of I-135

Compound I-135 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using4-(aminomethyl)pyridin-2-amine in place of methylamine in Step 5. MSm/z: 621.4 (M+H⁺).

Example 137: Synthesis of I-136

Compound I-136 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using(1-methyl-1H-pyrazol-3-yl)methanamine in place of methylamine in Step 5.MS m/z: 609.4 (M+H⁺).

Example 138: Synthesis of I-137

Compound I-137 was prepared as described in Example 21.7-chloro-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-((2-methylthiazol-4-yl)methyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-onewas used in place of Intermediate 2 and was prepared as described inExample 1 using (2-methylthiazol-4-yl)methanamine in place ofmethylamine in Step 5. MS m/z: 632.5 (M+H⁺).

Example 139: Synthesis of I-138 (Racemic)

Compound I-138 was prepared as described in Example 21 usingcis-1-methylpyrrolidine-3,4-diamine in place ofcis-cyclohexane-1,2-diamine in Step 2. MS m/z: 536.4 (M+H⁺).

Example 140: Synthesis of I-139 (Racemic)

Compound I-139 was prepared as described in Example 21 usingcis-1-(3,4-diaminopyrrolidin-1-yl)ethanone in place ofcis-cyclohexane-1,2-diamine in Step 2. MS m/z: 564.5 (M+H⁺).

Example 141: Synthesis of I-140

The title compound (2 mg) was prepared as described in Example 84 usingtert-butyl ((3R,4R)-4-aminotetrahydro-2H-pyran-3-yl)carbamate in placeof ((1S, 2R)-2-aminocyclohexyl)carbamate in Step 3, and starting with aderivative of Intermediate 6 from Example 1 (prepared using benzylaminein place of methylamine in Step 5). MS m/z: 537.2 (M+H⁺).

Example 142: Synthesis of I-141

Compound I-141 was prepared as described in Example 29 usingl-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole inplace of Intermediate 3 in Step 2 and Common Intermediate 6 from Example1 in Step 4. MS m/z: 609.4 (M+H⁺).

Example 143: Synthesis of I-142

Step 1: I-142

Intermediate 2 from Example 4 (43.0 mg, 90.5 μmol) was suspended in DCM(1 mL) and Et₃N (37.8 μl, 272 μmol) was added. The suspension was cooledto 0° C. and methacryloyl chloride (9.81 μl, 99.5 μmol) was added. Thereaction mixture was allowed to stir at 0° C. for 45 min, methanol wasadded at 0° C., and the reaction mixture was concentrated under reducedpressure. The resultant residue was purified through flashchromatography on silica gel (5% EtOAc in DCM) to afford 3.4 mg of thetitle compound. MS m/z: 543.5 (M+H⁺). ¹H NMR (400 MHz, DMSO-d6) δ 9.49(s, 1H), 8.71 (s, 1H), 8.11 (d, 1H), 7.74 (dd, 1H), 7.50 (dd, 1H), 7.17(ddd, 2H), 7.00 (s, 1H), 5.80 (m, 1H), 5.52 (t, 1H), 4.52 (s, 2H), 3.96(s, 6H), 3.22 (s, 3H), 1.94 (s, 3H).

Example 144: I-143

The title compound was prepared as described in Example 84 starting fromStep 2. Intermediate 2 was prepared as described in Example 117,Intermediate 7 substituting methylamine for 2,2-difluoroethanamine inStep 6. MS m/z: 503.1 (M+H⁺). ¹H NMR (400 MHz, CD₃OD): δ: 7.94 (1H, s),6.96 (1H, t), 6.29 (1H, br), 6.19 (1H, dd), 5.64 (1H, dd), 4.65 (2H, s),4.44 (2H, br), 3.92 (6H, s), 3.42 (3H, s), 1.78 (6H, br), 1.56 (2H, br).

Example 145: Synthesis of I-144

Compound I-144 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using tert-butyl(5-(aminomethyl)-2-fluorophenyl)carbamate in place of methylamine inStep 5. A final BOC deprotection step was performed (as described inExample 3, Step 5). MS m/z: 638.5 (M+H⁺).

Example 146: Synthesis of I-145

Compound I-145 was prepared as described in Example 17 using4-(2-methyl-2H-tetrazol-5-yl)-2-nitroaniline (Bioorganic & MedicinalChemistry Letters, 18(18), 4997-5001; 2008) in place of Intermediate 1,and skipping Step 2. MS m/z: 611.4 (M+H⁺).

Example 147: Synthesis of I-146

Compound I-146 was prepared as described in Example 17 using4-(2-methyl-2H-tetrazol-5-yl)-2-nitroaniline (Bioorganic & MedicinalChemistry Letters, 18(18)), in place of Intermediate 1, usingIntermediate 5 from Example 1, and skipping Step 2. MS m/z: 543.5(M+H⁺).

Example 148: Synthesis of I-147

Compound I-147 was prepared as described in Example 116. The startingmaterial was prepared as described in Example 1 using(1-methyl-1H-pyrazol-3-yl)methanamine in place of methylamine in Step 5.MS m/z: 615.5 (M+H⁺).

Example 149: Synthesis of I-148

Compound I-148 was prepared as described in Example 116. The startingmaterial was prepared as described in Example 1 using(1-methyl-1H-pyrazol-4-yl)methanamine in place of methylamine in Step 5.MS m/z: 605.6 (M+H⁺).

Example 150: Synthesis of I-149

Compound I-149 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using(1-methyl-1H-pyrazol-4-yl)methanamine in place of methylamine in Step 5.MS m/z: 522.6 (M+H⁺).

Example 151: Synthesis of I-150

Compound I-150 was prepared as described in Example 7. tert-butyl(2-amino-5-((1-methylpiperidin-4-yl)carbamoyl)phenyl)carbamate was usedin place of benzene-1,2-diamine in Step 1. Intermediate 6 from Example 1was used in place of Intermediate 5. MS m/z: 669.5 (M+H⁺).

Example 152: Synthesis of I-151

Compound I-151 was prepared as described in Example 7. tert-butyl(2-amino-5-((1-methylpiperidin-3-yl)carbamoyl)phenyl)carbamate was usedin place of benzene-1,2-diamine in Step 1. MS m/z: 601.5 (M+H⁺).

Example 153: Synthesis of I-152

Compound I-152 was prepared as described in Example 116. The startingmaterial was prepared as described in Example 1 using tert-butyl4-(aminomethyl)-1H-pyrazole-1-carboxylate in place of methylamine inStep 5. A final BOC deprotection step was performed as described inExample 3, Step 5. MS m/z: 601.4 (M+H⁺).

Example 154: Synthesis of I-153

Compound I-154 was prepared as described in Example 17 using4-(1-methyl-1H-tetrazol-5-yl)-2-nitroaniline (prepared as described inPCT Int. Appl., 2007066201, 14 Jun. 2007) in place of Intermediate 1,and skipping Step 2. MS m/z: 611.4 (M+H⁺).

Example 155: Synthesis of I-154 (Racemic)

Compound I-155 was prepared as described in Example 7.Cis-1-(1-methylpiperidin-4-yl)pyrrolidine-3,4-diamine was used in placeof benzene-1,2-diamine in Step 1. Intermediate 6 from Example 1 was usedin place of Intermediate 5. MS m/z: 547.1 (M+H⁺).

Example 156: I-155

Intermediate 7

The title compound was prepared according the literature (J. Med. Chem.2005, 48, 4628-4653) and as outlined in the scheme above.

The title compound was prepared form Intermediate 7 as outlined inExample 7 Step 2. MS m/z: 443.1 (M+H⁺). ¹H NMR (400 MHz, DMSO-d6): δ0.9.92 (1H, br s), 9.51 (1H, br s), 8.99 (1H, s), 8.15 (2H, br s),7.76-7.67 (2H, m), 7.26-7.26 (2H, m), 6.64-6.62 (4H, m), 6.62 (1H, dd),6.28 (1H, dd), 5.78 (1H, dd).

Example 157: Synthesis of I-156

Compound I-156 was prepared as described in Example 116. The startingmaterial was prepared as described in Example 1 using tert-butyl3-(aminomethyl)-1H-pyrazole-1-carboxylate in place of methylamine inStep 5. A final BOC deprotection step was performed as described inExample 3, Step 5. MS m/z: 601.4 (M+H⁺).

Example 158: Synthesis of I-157

Compound I-157 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using tert-butyl5-(aminomethyl)-1H-pyrazole-1-carboxylate in place of methylamine inStep 5. A final BOC deprotection step was performed as described inExample 3, Step 5. MS m/z: 595.4 (M+H⁺).

Example 159: Synthesis of I-158

Compound I-158 was prepared as described in Example 17 using4-(1-methyl-1H-tetrazol-5-yl)-2-nitroaniline (prepared as described inPCT Int. Appl., 2007066201, 14 Jun. 2007) in place of Intermediate 1,Intermediate 5 from Example 1 in place of Intermediate 6, and skippingStep 2. MS m/z: 543.5 (M+H⁺).

Example 160: Synthesis of I-159 (Racemic)

Compound I-159 was prepared as described in Example 7. cis-tert-butyl3,4-diaminopyrrolidine-1-carboxylate was used in place ofbenzene-1,2-diamine in Step 1. Intermediate 6 from Example 1 was used inplace of Intermediate 5. MS m/z: 622.5 (M+H⁺).

Example 161: Synthesis of I-160 (Racemic)

Compound I-160 was prepared as described in Example 7. tert-butyl(2-((cis)-3,4-diaminopyrrolidin-1-yl)-2-oxoethyl)carbamate was used inplace of benzene-1,2-diamine in Step 1. Intermediate 6 from Example 1was used in place of Intermediate 5. A final BOC deprotection step wasperformed as described in Example 3, Step 5. MS m/z: 579.5 (M+H⁺).

Example 162: Synthesis of I-161 (Racemic)

Compound I-161 was prepared as described in Example 7.1-((cis)-3,4-diaminopyrrolidin-1-yl)-2-hydroxyethanone was used in placeof benzene-1,2-diamine in Step 1. Intermediate 6 from Example 1 was usedin place of Intermediate 5. MS m/z: 580.4 (M+H⁺).

Example 163: Synthesis of I-162

Compound I-162 was prepared as described in Example 116 using tert-butyl((1S,2R)-2-aminocyclopentyl)carbamate in place of tert-butyl ((1S,2R)-2-aminocyclohexyl)carbamate in Step 2, and starting withIntermediate 6 from Example 1. MS m/z: 521.1 (M+H⁺).

Example 164: Synthesis of I-163

Compound I-163 was prepared as described in Example 7. The startingmaterial was prepared as described in Example 1 using tert-butyl5-(aminomethyl)-1H-imidazole-1-carboxylate in place of methylamine inStep 5. A final BOC deprotection step was performed as described inExample 3, Step 5. MS m/z: 595.4 (M+H⁺).

Example 165: Synthesis of I-164

Compound I-164 was prepared as described in Example 7.((cis)-3,4-diaminopyrrolidin-1-yl)(1-methylpiperidin-4-yl)methanone wasused in place of benzene-1,2-diamine in Step 1. Intermediate 6 fromExample 1 was used in place of Intermediate 5. MS m/z: 647.5 (M+H⁺).

Example 166: Synthesis of I-165

To a solution of Intermediate 10 from Example 178 (86.0 mg, 0.13 mmoL)in 2 mL DCM was added 200 uL of HCl (4 N in dioxane) and the reactionwas stirred at rt for 30 min. The solvent was removed under reducedpressure to obtain the amine hydrochloride salt. MS m/z: 553.2 (M+H⁺).To a solution of salt and acrylic acid (10 uL, 0.13 mmoL) in 500 uL ofDMF was cooled in ice-water/methanol bath. To the mixture was added DIEA(130 uL, 0.74 mmol) and then HATU (55 mg, 0.13 mmol). The reaction wasstirred at rt for 15 mins and directly purified by flash chromatography(eluting with a gradient of 0-70% acetone in heptane), which gave 36 mgof the title compound. MS m/z: 607.1 (M+H⁺).

Example 167: Synthesis of I-166 (Racemic)

Compound I-166 was prepared as described in Example 7.((cis)-3,4-diaminopyrrolidin-1-yl)(1-methylpiperidin-2-yl)methanone wasused in place of benzene-1,2-diamine in Step 1. Intermediate 6 fromExample 1 was used in place of Intermediate 5. MS m/z: 647.5 (M+H⁺).

Example 168: Synthesis of I-167 (Racemic)

Compound I-167 was prepared as described in Example 7.((cis)-3,4-diaminopyrrolidin-1-yl)(1-methylpiperidin-3-yl)methanone wasused in place of benzene-1,2-diamine in Step 1. Intermediate 6 fromExample 1 was used in place of Intermediate 5. MS m/z: 647.5 (M+H⁺).

Example 169: Synthesis of I-168

To a solution of I-165, from Example 166, (34.0 mg, 0.06 mmoL) in 500 uleach of IP A, THF, and water, was added LiOH mono-hydrate (7.50 mg, 0.18mmoL), and the reaction mixture was allowed to stir at ambienttemperature for 2 h. The solvent was removed under reduced pressure andthe resultant residue treated with 2 drops of 3 N HCl to causeprecipitation. The precipitate was filtered and dried to provide 14.0 mgof the title compound. MS m/z: 579.2 (M+H⁺).

Example 170: Synthesis of I-169

Compound I-169 was prepared as described in Example 116. The startingmaterial was prepared as described in Example 1 using3-methoxy-5-(trifluoromethyl)aniline in place of 3,5-dimethoxyaniline inStep 4 and skipping Step 7. MS m/z: 505.5 (M+H⁺).

Example 171: Synthesis of I-170 (Racemic)

Compound I-170 was prepared as described in Example 7.(cis)-1-(pyridin-2-yl)pyrrolidine-3,4-diamine was used in place ofbenzene-1,2-diamine in Step 1. Intermediate 6 from Example 1 was used inplace of Intermediate 5. MS m/z: 559.4 (M+H⁺).

Example 172: Synthesis of I-171

Compound I-171 was prepared as described in Example 116. The startingmaterial was prepared as described in Example 1 using tert-butyl5-(aminomethyl)-1H-imidazole-1-carboxylate in place of methylamine inStep 5. A final BOC deprotection step was performed as described inExample 3, Step 5. MS m/z: 601.4 (M+H⁺).

Example 173: Synthesis of I-172 (Racemic)

Compound I-172 was prepared as described in Example 174 skipping Step 4.MS m/z: 463.2 (M+H⁺).

Example 174: Synthesis of I-173

Steps 1-2: Intermediate 2

A mixture of Intermediate 1 (WO 2009153313) (102 mg, 0.37 mmol) andcis-1,2-diaminocylcohexane (0.20 mL, 1.67 mmol) in dioxane (2.0 mL) washeated at 110° C. for 16 h. The reaction mixture was allowed to cool toambient temperature, concentrated to dryness, dissolved in DCM (20 mL),and treated with (BocfiO (1.20 g) at ambient temperature, then allowedto stir overnight. The crude reaction was then concentrated andsubjected to flash chromatography on silica gel (0-100% EtOAc/hexanes)to afford 50 mg for the title compound. MS: 452.2 [M+H]⁺

Step 3: Intermediate 3

Intermediate 2 (50.0 mg, 0.110 mmol) and2-(3,5-dimethoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (60.0mg, 0.22 mmol), and Pd(dppf)Cl₂ (10.0 mg, 0.014 mmol) were combined indioxane (3 mL) and 2.0 M aqueous Na₂CO₃ (0.7 mL). The mixture was heatedat 120° C. for 30 min in a microwave reactor. The reaction mixture wasdiluted with EtOAc, washed with saturated aqueous NaHCO₃, brine, driedover Na₂SO₄, filtered and concentrated in vacuo. The resultant residuewas purified by flash chromatography on silica gel to provide 48 mg ofthe title compound. MS m/z: 509.4 (M+H⁺).

Steps 4-5: Intermediate 4

Intermediate 3 (40.0 mg, 0.079 mmol) was dissolved in DCM (2 mL) andMeCN (2 mL). At 0° C., SO₂Cl₂ (13 μL, 0.16 mmol) was added and thereaction was kept at 0° C. for 2.5 hr. After removing solvents, thecrude mixture was treated with 20% TFA/DCM (4.0 mL) at room temperaturefor 30 min. After evaporating all volatiles, the residue was taken up inDCM and treated with silica supported carbonate to free the basic amine.After filtration and concentration, quantitative amount of product wasobtained and the crude was used as-is in the next step. MS m/z: 477.4(M+H⁺).

Step 6: I-173

Intermediate 4 (40 mg, 0.088 mmol) was dissolved in THF (3.0 mL) andcooled to −10° C. Acryloyl chloride (7.2 μL, 0.088 mmol) was added andthe reaction mixture was allowed to stir at -10° C. for 10 min. Thereaction mixture was concentrated and the resultant residue dissolved inDMSO and purified by prep-HPLC. MS m/z: 531.2 (M+H⁺). 1H NMR (400 MHz,CD3OD): 9.25 (1H, s), 8.15 (1H, s), 7.77 (1H, s), 6.91 (1H, s), 6.36(1H, dd), 6.16 (1H, dd), 5.62 (1H, dd), 4.56 (2H, m), 3.97 (6H, s), 3.95(3H, s), 1.86 (6H, m), 1.60 (2H, m).

Example 175: Synthesis of I-174 (Racemic)

Compound I-174 was prepared as described in Example 7. tert-butyl((trans)-4-amino-6-oxopiperidin-3-yl)carbamate was used in place ofbenzene-1,2-diamine in Step 1. Intermediate 6 from Example 1 was used inplace of Intermediate 5. A final BOC deprotection step was performed asdescribed in Example 3, Step 5. MS m/z: 550.4 (M+H⁺).

Example 176: Synthesis of I-175

The title compound was prepared as outlined in Example 102 where thechloro pyrimidine cyclic urea intermediate (equivalent of Intermediate 7in Example 102) was prepared as described in Example 102 using2-chloro-6-fluoro-3,5-dimethoxyaniline in place of Intermediate 4,methylamine in place of cyclopropylmethanamine in Step 5, and tert-butyl((1S,2R)-2-aminocyclohexyl)carbamate in place of 1, 2-benzenediamine inStep 7. MS m/z: 519.4 (M+H)⁺. ¹H NMR (400 MHz, Chloroform-d) δ 7.81 (s,1H), 6.63 (d, 1H), 6.23 (ddd, 1H), 6.03 (ddd, 1H), 5.59 (ddd, 1H), 4.52(d, 2H), 4.41 (s, 1H), 4.17 (d, 1H), 3.92 (d, 6H), 3.38 (s, 3H),2.00-1.00 (m, 9H).

Example 177: Synthesis of I-176

Compound I-176 was prepared as described in Example 116. The startingmaterial was prepared as described in Example 1 using 4-methoxyanilinein place of 3,5-dimethoxyaniline in Step 4 and skipping Step 7. MS m/z:437.4 (M+H⁺).

Example 178: Synthesis of I-177

Step 1: Intermediate 10

The title compound was prepared according to the literature and asoutlined in the scheme above. A solution of Intermediate 8 (US2005/0020645 A1; Bioorganic & Medicinal Chemistry, 2009, 17, 1193-1206)(45.0 mg, 0.16 mmoL), Intermediate 9, prepared in a manner as describedin Example 116, using Intermediate 6 from Example 1, (45 mg, 0.08 mmoL),NMM (26 uL, 0.24 mmoL) in 1 mL of dioxane, and 100 uL of NMP, was heatedto 100° C. for 16 h. The solvent was removed under reduced pressure andthe resultant residue was purified by flash chromatography on silica gel(eluting with a gradient of 0-100% EtOAc in heptane), which gave 40 mgof the title compound. MS m/z: 653.2 (M+H⁺).

Step 2: Intermediate 11

To a solution of the Intermediate 10 (100 mg, 0.15 mmoL) in 1 mL of THFand 500 uL of water was added LiOH mono-hydrate (20.0 mg, 0.46 mmoL),and the reaction was stirred at 60° C. for 2 h. The solvent was removedunder reduced pressure and the resultant residue treated with 5 drops of3 N HCl to cause precipitation. The precipitate was filtered and driedwhich gave 50 mg of the title compound. MS m/z: 625.1 (M+H⁺).

Step 3: Intermediate 12

To a solution of the Intermediate 11 (50 mg, 0.08 mmoL) in 500 uL of THFwas added HATU (30.0 mg, 0.08 mmoL), NMM (26 uL, 0.24 mmoL) and excessethylamine (2 M in THF, 5 mL). The reaction mixture was allowed to stirat ambient temperature for 5 days. The solvent was removed under reducedpressure and the resultant residue purified by flash chromatography onsilica gel (eluting with a gradient of 0-100% acetone in heptane) whichgave 25.0 mg of the title compound. MS m/z: 652.2 (M+H⁺).

Steps 4 & 5: I-177

To a solution of the Intermediate 12 (25 mg, 0.04 mmoL) in 5 mL of DCMwas added 1 mL of HCl (4 N in dioxane) and the reaction was allowed tostir at ambient temperature for 30 min. The solvent was removed underreduced pressure to obtain the amine hydrochloride salt of Intermediate12, MS m/z: 552.2 (M+H⁺), which was taken up in 500 uL of DMF andacrylic acid (2.4 ul, 0.04 mmoL) was added. The reaction mixture wascooled in ice-water/methanol bath and DIPEA was added (40 uL, 0.21 mmoL)followed by HATU (14.0 mg, 0.04 mmol). The reaction mixture was allowedto stir at ambient temperature for 15 min then purified by preparativeHPLC (eluting with a gradient of 10-90% water containing 0.1% TFA andMeCN), which gave 7.0 mg of the title compound. MS m/z: 606.2 (M+H⁺).

Example 180: Synthesis of I-179

The title compound was prepared as described in Example 3. Tert-butyl3-aminobenzylcarbamate was used in place of benzene-1,2-diamine in Step1, and the Boc protection of Step 2 was skipped. MS m/z: 690.2 (M+H⁺).

Example 181: Synthesis of I-180

The title compound was prepared as described in Example 3. Tert-butyl3-aminobenzylcarbamate was used in place of benzene-1,2-diamine inStep 1. A BOC deprotection step was performed prior to Step 2 (asdescribed in Example 3, Step 5). MS m/z: 690.1 (M+H⁺).

Example 182: Synthesis of I-181

The title compound was prepared as described in Example 7. Tert-butyl3-aminobenzylcarbamate was used in place of benzene-1,2-diamine inStep 1. Intermediate 6 from Example 1 was used in place of Intermediate5. A BOC deprotection step was performed prior to Step 2 (Bocdeprotection is described in Example 3, Step 5). MS m/z: 543.3 (M+H⁺).

Example 183: Synthesis of I-182 (Racemic)

The title compound was prepared as described in Example 7.4,5-dimethoxycyclohexane-cis-1,2-diamine was used in place ofbenzene-1,2-diamine in Step 1. Intermediate 6 from Example 1 was used inplace of Intermediate 5. MS m/z: 595.5 (M+H⁺).

Example 184: Synthesis of I-183 (Racemic)

The title compound was prepared as described in Example 7.Cis-1-(pyridazin-3-yl)pyrrolidine-3,4-diamine was used in place ofbenzene-1,2-diamine in Step 1. Intermediate 6 from Example 1 was used inplace of Intermediate 5. MS m/z: 600.4 (M+H⁺).

Example 185: Synthesis of I-184 (Racemic)

The title compound was prepared as described in Example 116. Thestarting material was prepared as described in Example 1 using2-chloro-6-fluoro-3,5-dimethoxyaniline in place of 3,5-dimethoxyanilinein Step 4 and skipping Step 7. MS m/z: 519.4 (M+H⁺).

Example 186: Chiral Separation of I-185 and I-186

Chiral separation of I-126, (Chiralpak IA, 250 mm×4.6 mm ID, 5 micron,0.4 mL/min, 30% heptane in EtOH) provided two enantiomers with Rt=23 and31 min which were assigned absolute configurations of I-186 and I-185,respectively (>98% ee). Absolute configurations were assigned by analogyto I-94 and I-95 based on enzymatic and cellular potency.

I-185: MS m/z: 523.5 (M+H⁺), [α]_(D)=−20 (C=1.00 mg/mL, CH₂Cl₂, 23° C.),¹H NMR (400 MHz, CDCl₃) δ: 3.39 (s, 3H), 3.71-3.81 (m, 2H), 3.94 (s,6H), 4.17-4.21 (m, 2H), 4.52 (s, 2H), 4.73-4.79 (m, 2H), 5.62-5.65 (m,2H), 6.02-6.07 (m, 1H), 6.26 (d, 1H), 6.32 (br, 1H), 6.60 (s, 1H), 7.84(s, 1H).

I-186: MS m/z: 523.5 (M+H⁺), [α]_(D)=+38 (C=1.00 mg/mL, CH₂Cl₂, 23° C.),¹H NMR (400 MHz, CDCl₃) δ: 3.39 (s, 3H), 3.71-3.81 (m, 2H), 3.94 (s,6H), 4.17-4.21 (m, 2H), 4.52 (s, 2H), 4.73-4.79 (m, 2H), 5.62-5.65 (m,2H), 6.02-6.07 (m, 1H), 6.26 (d, 1H), 6.32 (br, 1H), 6.60 (s, 1H), 7.84(s, 1H).

The absolute configuration of I-186 was confirmed throughenantioselective synthesis according to the below scheme.N-((3R,4S)-4-aminotetrahydrofuran-3-yl)acrylamide was prepared accordingto literature (JACS, 1995, 117, 5897-5898) and as described in Example226, and used in place of cis-tetrahydrofuran-3,4-diamine in Example127.

Steroechemical Proof and Enantioselective Synthesis of I-186

Intermediate 7 (scheme above) was synthesized according to the proceduredescribed in Example 226. Intermediate 7 was was used to make I-186, bycoupling with Intermediate 6 from Example 127 followed by Bocdeprotection and acrylamide formation using the procedure described inExample 127. I-186: MS m/z: 523.5 (M+H⁺), NMR (400 MHz, CDCl₃) δ: 3.39(s, 3H), 3.71-3.81 (m, 2H), 3.94 (s, 6H), 4.17-4.21 (m, 2H), 4.52 (s,2H), 4.73-4.79 (m, 2H), 5.65 (dd, 1H), 6.02-6.07 (m, 1H), 6.26 (d, 1H),6.32 (br, 1H), 6.60 (s, 1H), 7.84 (s, 1H).

Example 187: Synthesis

Using the techniques described herein, the following compounds can beprepared. For compounds prepared as racemic or diastereomeric mixtures,the single isomers can be prepared in optically pure form by eitheremploying chiral starting materials or performing chiral chromatography.

Compound I-187

The title compound is prepared as described in Example 7. The startingmaterial is prepared as described in Example 1 using4-aminobicyclo[2.2.2]octan-1-ol in place of 3,5-dimethoxyaniline in Step4 and skipping Step 7.

Compound I-188

The title compound is prepared as described in Example 7. The startingmaterial is prepared as described in Example 1 using4-aminobicyclo[2.2.2]octan-1-ol in place of 3,5-dimethoxyaniline in Step4, benzylamine in place of methylamine in Step 5, and skipping Step 7.

Compound I-189

The title compound is prepared as described in Example 7. The startingmaterial is prepared as described in Example 1 using4-aminobicyclo[2.2.2]octan-1-ol in place of 3,5-dimethoxyaniline in Step4, cyclopropanamine in place of methylamine in Step 5, and skipping Step7.

Compound I-190

The title compound is prepared as described in Example 116. The startingmaterial is prepared as described in Example 1 using3-(aminomethyl)benzenesulfonamide in place of methylamine in Step 5.

Compound I-191

The title compound is prepared as described in Example 7 usingtert-butyl tert-butyl(2-amino-5-(4-ethylpiperazin-1-yl)cyclohexyl)carbamate in place ofbenzene-1,2-diamine in Step 1, Intermediate 6 from Example 1 in place ofIntermediate 5, and adding a final BOC deprotection step as described inExample 3, Step 5.

Compound I-193

The title compound is prepared as described in Example 7 usingtert-butyl ((cis)-5-amino-2-oxopiperidin-4-yl)carbamate in place ofbenzene-1,2-diamine in Step 1, Intermediate 6 from Example 1 in place ofIntermediate 5, and adding a final BOC deprotection step as described inExample 3, Step 5.

Compound I-194

The title compound is prepared as described in Example 7 usingtert-butyl ((cis)-4-amino-6-oxopiperidin-3-yl)carbamate in place ofbenzene-1,2-diamine in Step 1, Intermediate 6 from Example 1 in place ofIntermediate 5, and adding a final BOC deprotection step as described inExample 3, Step 5.

Compound I-195

The title compound is prepared as described in Example 7 usingtert-butyl ((trans)-5-amino-2-oxopiperidin-4-yl)carbamate in place ofbenzene-1,2-diamine in Step 1, Intermediate 6 from Example 1 in place ofIntermediate 5, and adding a final BOC deprotection step as described inExample 3, Step 5.

Example 188: Synthesis of I-196

The title compound was prepared as described in Example 7 usingtert-butyl ((trans)-4-amino-6-oxopiperidin-3-yl)carbamate in place ofbenzene-1,2-diamine in Step 1, Intermediate 6 from Example 1 in place ofIntermediate 5, and adding a final BOC deprotection step as described inExample 3, Step 5. MS m/z: 550.4 (M+H⁺).

Example 189: Synthesis

Using the techniques described herein, the following compounds can beprepared. For compounds prepared as racemic or diastereomeric mixtures,the single isomers can be prepared in optically pure form by eitheremploying chiral starting materials or performing chiral chromatography.

Compound I-197

The title compound is prepared as described in Example 127. The chlorocyclic urea derivative is prepared as described in Example 1 using3,5-dimethoxy-2,6-dimethylaniline in place of 3,5-dimethoxyaniline inStep 4, and skipping Step 7.

Compound I-198

The title compound was prepared as described in Example 127. The chlorocyclic urea derivative was prepared as described in Example 1 using2,6-difluoro-3,5-dimethoxyaniline in place of 3,5-dimethoxyaniline inStep 4 and skipping Step 7. MS m/z: 491.5 (M+H⁺).

Compound I-199

The title compound is prepared as described in Example 116. The startingmaterial is prepared as described in Example 1 using2,6-dimethyl-3,5-dimethoxyaniline in place of 3,5-dimethoxyaniline inStep 4 and skipping Step 7.

Compound I-200

The title compound was prepared as described in Example 127 using2,5-dihydrothiophene in place of Intermediate 2 and oxidation of sulfurwas performed as described in the literature (JOC, 2010, 75, 4629-4631).MS m/z: 571.4 (M+H⁺).

Compound I-201

The title compound was prepared as described in Example 7 using(cis)-1-(pyrimidin-4-yl)pyrrolidine-3,4-diamine in place ofbenzene-1,2-diamine in Step 1, and Intermediate 6 from Example 1 inplace of Intermediate 5. MS m/z: 600.5 (M+H⁺).

Example 190: Synthesis of I-202

The title compound was prepared as described in Example 7 using(cis)-1-(pyridin-2-yl)pyrrolidine-3,4-diamine in place ofbenzene-1,2-diamine in Step 1, and Intermediate 6 from Example 1 inplace of Intermediate 5. MS m/z: 599.4 (M+H⁺).

Example 191: Synthesis

Using the techniques described herein, the following compounds can beprepared. For compounds prepared as racemic or diastereomeric mixtures,the single isomers can be prepared in optically pure form by eitheremploying chiral starting materials or performing chiral chromatography.

Compound I-203

The title compound is prepared as described in Example 7 using(cis)-1-(1H-pyrazol-3-yl)pyrrolidine-3,4-diamine in place ofbenzene-1,2-diamine in Step 1 and Intermediate 6 from Example 1 in placeof Intermediate 5.

Example 192: Synthesis of I-204

The title compound was prepared as described in Example 7 using(cis)-1-(1-methyl-1H-pyrazol-3-yl)pyrrolidine-3,4-diamine in place ofbenzene-1,2-diamine in Step 1 and Intermediate 6 from Example 1 in placeof Intermediate 5. MS m/z: 602.5 (M+H⁺).

Example 193: Synthesis of I-205

The title compound was prepared as described in Example 127. The chlorocyclic urea derivative was prepared as described in Example 1 using3,5-diethoxyaniline in place of 3,5-dimethoxyaniline in Step 4. MS m/z:551.5 (M+H⁺).

Example 194: Synthesis of I-206

The title compound was prepared as described in Example 127. The chlorocyclic urea derivative was prepared as described in Example 1 using2-chloro-6-fluoro-3,5-dimethoxyaniline in place of 3,5-dimethoxyanilinein Step 4 and skipping Step 7. MS m/z: 507.4 (M+H⁺).

Example 195: Synthesis of I-207

The title compound was prepared as described in Example 127. The chlorocyclic urea derivative was prepared as described in Example 1 using3,4,5-trimethoxyaniline in place of 3.5-dimethoxyaniline in Step 4 andskipping Step 7. MS m/z: 485.5 (M+H⁺).

Example 196: Synthesis of I-208

The title compound was prepared as described in Example 127. The chlorocyclic urea derivative was prepared as described in Example 1 using3,4,5-trimethoxyaniline in place of 3.5-dimethoxyaniline in Step 4. MSm/z: 553.4 (M+H⁺).

Example 197: Synthesis of I-209

The title compound was prepared as described in Example 127. The chlorocyclic urea derivative was prepared as described in Example 1 using4-methoxyaniline in place of 3,5-dimethoxyaniline in Step 4 and skippingStep 7. MS m/z: 425.4 (M+H⁺).

Example 198: Synthesis of I-210

The title compound was prepared as described in Example 127. The chlorocyclic urea derivative was prepared as described in Example 1 usingthiazol-4-ylmethanamine in place of methylamine in Step 5. MS m/z: 606.4(M+H⁺).

Example 199: Synthesis of I-212

The title compound was prepared as described in Example 127. The chlorocyclic urea derivative was prepared as described in Example 1 usingcyclopropylmethanamine in place of methylamine in Step 5. MS m/z: 563.4(M+H⁺).

Example 200: Synthesis of I-213

The title compound was prepared as described in Example 127. The chlorocyclic urea derivative was prepared as described in Example 1 using(1-methyl-1H-pyrazol-4-yl)methanamine in place of methylamine in Step 5.MS m/z: 603.5 (M+H⁺).

Example 201: Synthesis of I-214

The title compound was prepared as described in Example 127. The chlorocyclic urea derivative is prepared as described in Example 1 using(1H-pyrazol-4-yl)methanamine in place of methylamine in Step 5. MS m/z:589.5 (M+H⁺).

Example 202: Synthesis of I-215

The title compound was prepared as described in Example 127. The chlorocyclic urea derivative is prepared as described in Example 1 using(1-methyl-1H-pyrazol-3-yl)methanamine in place of methylamine in Step 5.MS m/z: 603.5 (M+H⁺).

Example 203: Synthesis of I-216

The title compound was prepared as described in Example 127. The chlorocyclic urea derivative is prepared as described in Example 1 using(1H-pyrazol-3-yl)methanamine in place of methylamine in Step 5. MS m/z:589.5 (M+H⁺).

Example 204: Synthesis of I-217

The title compound was prepared as described in Example 127. The chlorocyclic urea derivative is prepared as described in Example 1 usingethylamine in place of methylamine in Step 5. MS m/z: 537.4 (M+H⁺).

The title compound was prepared as described in Example 127 with amethylation step (as described in Example 31, Step 1) prior to Step 5.MS m/z: 537.5 (M+H⁺).

Example 206: Synthesis of I-219

The title compound was prepared as described in Example 116. Thestarting material was prepared as described in Example 1 using3,5-diethoxyaniline in place of 3,5-dimethoxyaniline in Step 4. MS m/z:563.5 (M+H⁺).

Example 207: Synthesis of I-220

The title compound was prepared as described in Example 116. Thestarting material was prepared as described in Example 1 using 3,4,5trimethoxyaniline in place of 3,5-dimethoxyaniline in Step 4 andskipping Step 7. MS m/z: 497.5 (M+H⁺).

Example 208: Synthesis

Using the techniques described herein, the following compounds can beprepared. For compounds prepared as racemic or diastereomeric mixtures,the single isomers can be prepared in optically pure form by eitheremploying chiral starting materials or performing chiral chromatography.

Compound I-221

The title compound was prepared as described in Example 116. Thestarting material was prepared as described in Example 1 using 3,4,5trimethoxyaniline in place of 3,5-dimethoxyaniline in Step 4. MS m/z:565.5 (M+H⁺).

Example 209: Synthesis of I-222

The title compound was prepared as described in Example 116. Thestarting material was prepared as described in Example 1 using4-methoxyaniline in place of 3,5-dimethoxyaniline in Step 4, andskipping Step 7. MS m/z: 437.5 (M+H⁺).

Example 210: Synthesis of I-223

The title compound was prepared as described in Example 224 usingtert-butyl ((1S,2R)-2-aminocyclohexyl)carbamate in place ofcis-tetrahydrofuran-3,4-diamine in Step 1. MS m/z: 532.5 (M+H⁺). ¹H NMR(400 MHz, CDCl3): 1.50-1.78 (m, 8H), 3.68 (s, 3H), 3.95 (s, 6H), 4.30(br, 1H), 4.53 (br, 1H), 5.61 (d, 1H), 5.99-6.05 (m, 1H), 6.26 (d, 1H),6.63 (s, 1H), 7.41 (s, 1H), 8.44 (s, 1H).

Example 211: Synthesis

Using the techniques described herein, the following compounds can beprepared. For compounds prepared as racemic or diastereomeric mixtures,the single isomers can be prepared in optically pure form by eitheremploying chiral starting materials or performing chiral chromatography.

Compound I-224

The title compound is prepared as described in Example 116. The startingmaterial is prepared as described in Example 1 using3,5-bis(trifluoromethoxy)aniline in place of 3,5-dimethoxyaniline inStep 4.

Compound I-225

The title compound is prepared as described in Example 127. The chlorocyclic urea derivative is prepared as described in Example 1 using3,5-bis(trifluoromethoxy)aniline in place of 3,5-dimethoxyaniline inStep 4.

Compound I-226

The title compound is prepared as described in Example 7 using3,4-diaminocyclobut-3-ene-1,2-dione in place of benzene-1,2-diamine inStep 1 and Intermediate 6 from Example 1 in place of Intermediate 5.

Compound I-227

The title compound is prepared as described in Example 7. The startingmaterial is prepared as described in Example 1 using2-bromo-6-fluoro-3,5-dimethoxyaniline in place of 3,5-dimethoxyanilinein Step 4, cyclopropylmethanamine in place of methylamine in Step 5, andskipping Step 7.

Example 212: Synthesis of I-228

The title compound was prepared as described in Example 116. Thestarting material was prepared as described in Example 1 using2-bromo-6-fluoro-3,5-dimethoxyaniline in place of 3,5-dimethoxyanilinein Step 4 and skipping Step 7. MS m/z: 563.4 (M+H⁺).

Example 213: Synthesis

Using the techniques described herein, the following compounds can beprepared. For compounds prepared as racemic or diastereomeric mixtures,the single isomers can be prepared in optically pure form by eitheremploying chiral starting materials or performing chiral chromatography.

Compound I-229

The title compound is prepared as described in Example 116. The startingmaterial is prepared as described in Example 1 using2,4,6-trifluoro-3,5-dimethoxyaniline in place of 3,5-dimethoxyaniline inStep 4 and skipping Step 7.

Compound 363 I-230

The title compound is prepared as described in Example 116. The startingmaterial is prepared as described in Example 1 using2-cyclopropyl-6-fluoro-3,5-dimethoxyaniline in place of3,5-dimethoxyaniline in Step 4 and skipping Step 7.

Compound I-231

The title compound is prepared as described in Example 116. The startingmaterial is prepared as described in Example 1 using2-cyclopropyl-6-chloro-3,5-dimethoxyaniline in place of3,5-dimethoxyaniline in Step 4 and skipping Step 7.

Example 214: Synthesis of I-118

Compound I-118 was prepared as described in Example 116 using propionicacid in place of acrylic acid in Step 4. MS m/z: 602.2 (M+H⁺).

Example 215: Synthesis of I-232

Compound I-232 was prepared as described in Example 3. Tert-butyl(3-aminophenyl)carbamate was used in place of benzene-1,2-diamine inStep 1. MS m/z: 676.4 (M+H⁺).

Example 216: Synthesis of I-233

Compound I-233 was prepared as described in Example 3. Tert-butyl(4-aminophenyl)carbamate was used in place of benzene-1,2-diamine inStep 1. MS m/z: 676.3 (M+H⁺).

Example 217: Synthesis of I-234

Compound I-234 was prepared as described in Example 3. Propionylchloride was used in place of acryloyl chloride in Step 6. MS m/z: 678.2(M+H⁺).

Example 218: Synthesis of I-235

Compound I-235 was prepared as described in Example 7. Intermediate 6from Example 1 was used in place of Intermediate 5. Propionyl chloridewas used in place of acryloyl chloride in Step 2. MS m/z: 531.0 (M+H⁺).

Example 219: Synthesis of I-236

Compound I-236 was prepared as described in Example 5. Propionylchloride was used in place of acryloyl chloride in Step 2. MS m/z: 607.1(M+H⁺).

Example 220: Synthesis of I-237

Compound I-237 was prepared as described in Example 7. Aniline was usedin place of in place of benzene-1,2-diamine in Step 1. MS m/z: 392.3(M+H⁺).

Example 221: Synthesis of I-238

Compound I-238 was prepared as described in Example 7. Aniline was usedin place of benzene-1,2-diamine in Step 1. Intermediate 6 from Example 1was used in place of Intermediate 5. MS m/z: 460.1 (M+H⁺).

Example 222: Synthesis of I-239

To a solution of the intermediate I-186 (3.80 mg, 0.007 mmoL) in 500 uLof THF was added catalytic 10% Pd/C. 1 atm of H₂ was introduced viaballoon and the reaction mixture was allowed to stir at ambienttemperature for 1 h. The reaction mixture was filtered, through a pad ofcelite and the solvent was removed under reduced pressure to afford 3.7mg of the title compound. MS m/z: 525.2 (M+H⁺).

Example 223: Synthesis of Common Intermediate 8

Step 1: Intermediate 2

To a mixture of Intermediate 1 (1.35 g, 6.88 mmol) in EtOH was addedcone. H₂SO₄ (4 drops). The reaction mixture was heated at 85° C. for 16h after which it was cooled to ambient temperature and concentrated. Theresultant residue was diluted with water (20 mL) and extracted with DCM(25 mL×3). The combined organic layers were washed with brine (50 mL),dried over Na₂SO₄ and concentrated to afford the title compound (2.85 g,100%) as a yellow solid.

Step 2: Intermediate 4

Methylamine in EtOH (33%, 17.5 mL, 140 mmol) was slowly added to asolution of Intermediate 3 (10.0 g, 43.1 mmol) in 120 mL ofdichloromethane at 0° C. The solution was stirred for 30 min. Water (150ml) was added, and the resultant mixture was separated, the organiclayer was dried over MgSO₄, filtered, and concentrated to afford thetitle compound (9.77 g, 100%) as white solid.

Step 3: Intermediate 5

To a mixture of LAH (2.45 g, 64.6 mmol) in anhydrous THF (30 mL) at 0°C. a solution of Intermediate 4 (9.77 g, 43.0 mmol) in anhydrous THF (45mL) was added dropwise. The reaction mixture was allowed to stir for 15min at ambient temperature. Water (18 mL) was added dropwise withcaution. The mixture was stirred for 30 min. Aqueous NaOH solution (15%,8.5 mL) was added dropwise, followed by addition of water (26 mL). Theresulting suspension was allowed to stir for 17 h at ambient temperatureafter which the reaction mixture was filtered and sequentially washedwith THF (100 mL×2). The combined filtrate and washings wereconcentrated and the resultant residue was suspended in ethylacetate/hexane (v/v: 2:1, 200 mL). Solids were collected by filtrationto afford the title compound as yellow solid (4.23 g, 53%).

Step 4: Intermediate 6

Compound 5 (4.23 g, 23.2 mmol) was taken into dichloromethane (1 L) andtreated with manganese dioxide (18.0 g, 207 mmol) with stirring. Theresultant suspension was stirred for 24 h, then filtered through Celite,washed with dichloromethane (100 mL), and the combined organic layerswere concentrated to afford the title compound (3.00 g, 75%).

Step 5: Intermediate 7

A mixture of Intermediate 2 (1.29 g, 5.75 mmol), Intermediate 6 (1.00 g,5.46 mmol) and K₂CO₃ (1.50 g, 10.9 mmol) in DMF (100 mL) was heated to110° C. for 4 h. The mixture was allowed to cool to ambient temperature,poured into water, filtered, and the solids were dried to afford thetitle compound (1.20 g, 63%) as white solid.

Step 6: Intermediate 8

To a solution of Intermediate 7 (1.33 g, 3.88 mmol) in DCM (15 mL) andNMP (5 mL), was added SO₂Cl₂ (2.10 g, 15.6 mmol) dropwise at 0° C. Theresultant mixture was allowed to stir at 0° C. for 30 min after which itwas concentrated, diluted with water, extracted with EtOAc (25 mL×4) andthe combined organic layers were dried over Na₂SO₄. The organic layerswere concentrated to afford the title compound (1.50 g, 87%) as a whitesolid. The resultant solid was recrystallized with ethyl acetate orpurified by silica gel column chromatography to afford the titlecompound as white solid (1.20 g, 70%).

Example 224: Synthesis of I-211

Step 1: Intermediate 1

A mixture of Common Intermediate 8 from Example 223 (300 mg, 0.68 mmol),cis-tetrahydrofuran-3,4-diamine (204 mg, 2.0 mmol), and DIPEA (387 mg,3.0 mmol) in NMP (5 mL) was heated to 80° C. for 3 h. The mixture wasallowed to cool to ambient temperature and partitioned between EtOAc andwater. The organic phase was separated, washed with water, brine, driedover anhydrous Na₂SO₄ and the resultant residue was purified throughcolumn chromatography on silica to afford the title compound (137 mg,44%). MS m/z: 466.3 (M+H⁺).

Step 2: I-211

To a solution of Intermediate 1 (3.50 g, 7.5 mmol), DIPEA (1.94 g, 15mmol) in anhydrous DCM (100 mL) at 0° C. was added a solution ofacryloyl chloride (680 mg, 7.50 mmol) in anhydrous DCM (5 mL) dropwise.The reaction mixture was allowed to stir for 10 min after which waspartitioned between DCM and H₂O. The organic phase was separated, washedwith brine, dried over Na₂SO₄ and the crude product was purified throughsilica gel column chromatography to afford the title compound (2.90 g,74%). MS m/z: 520.4 (M+H⁺). ¹H NMR (400 Hz, CDCl₃): 3.68-3.85 (m, 5H),3.95 (s, 6H), 4.16-4.24 (m, 2H), 4.87 (br, 2H), 5.62 (d, 1H), 6.02-6.08(m, 1H), 6.24-6.39 (m, 3H), 6.63 (s, 1H), 7.43 (s, 1H), 8.47 (s, 1H).

Example 225: Chiral Separation of I-211 to afford I-240 and I-241

Chiral SFC separation of I-211 (ChiralCel OD-3, 150×4.6 mm, 5 micron,2.4 mL/min, 40% MeOH with 0.05% DEA in CO₂) provided two enantiomerswith Rt=3.46 and 4.89 min which were assigned absolute configurations ofI-240 and I-241 respectively (>98% ee). Absolute configurations wereassigned by analogy to I-94 and I-95, based on enzymatic and cellularpotency.

I-241: MS m/z: 520.4 (M+H⁺), [α]_(D)=+75 (C=4.00 mg/mL, CH₂Cl₂, 23° C.),¹H NMR (400 Hz, CDCl₃): 3.68-3.85 (m, 5H), 3.95 (s, 6H), 4.16-4.24 (m,2H), 4.87 (br, 2H), 5.62 (d, 1H), 6.02-6.08 (m, 1H), 6.24-6.39 (m, 3H),6.63 (s, 1H), 7.43 (s, 1H), 8.47 (s, 1H).

I-240: MS m/z: 520.4 (M+H⁺), [α]_(D)=−65 (C=4.00 mg/mL, CH₂Cl₂, 23° C.),¹H NMR (400 Hz, CDCl₃): 3.68-3.85 (m, 5H), 3.95 (s, 6H), 4.16-4.24 (m,2H), 4.87 (br, 2H), 5.62 (d, 1H), 6.02-6.08 (m, 1H), 6.24-6.39 (m, 3H),6.63 (s, 1H), 7.43 (s, 1H), 8.47 (s, 1H).

The absolute configuration of I-241 was confirmed throughenantioselective synthesis according to the below scheme.N-((3R,4S)-4-aminotetrahydrofuran-3-yl)acrylamide was prepared accordingto literature QIACS, 1995, II7, 5897-5898) and as described in Example226, and used in place of cis-tetrahydrofuran-3,4-diamine in Example224.

Example 226: Steroechemical Proof and Enantioselective Synthesis ofI-241

Step 1: Intermediate 2,((3R,4S)-4-azidotetrahydrofuran-3-yloxy)trimethylsilane

A flask equipped with a stir bar was charged with (R, R)-Salen catalyst(600 mg, 0.02 equiv, Sigma Aldrich, catalog 531944, CAS #164931-83-3),and flushed with N₂. Cyclopentene oxide (4.30 g, 50.0 mmol) and TMSN₃(6.00 g, 1.05 equiv) were added sequentially at ambient temperature. Thereaction mixture was allowed to stir 12 h, at which time excess TMSN₃was removed under reduced pressure, and the residue was purified bysilica gel column chromatography (eluting with 30% EtOAc/hexane) toafford the title compound as a yellow oil (7.80 g, 78%, 91% ee, JACS,1995, II7, 5897-5898). ¹H NMR (CDCl₃, 400 MHz): δ 0.00 (s, 9H),3.46-3.49 (m, 1H), 3.64-3.69 (m, 2H), 3.83 (dd, 1H), 3.89 (dd, 1H),4.08-4.11 (m, 1H).

Step 2: Intermediate 3, (3R,4S)-4-aminotetrahydrofuran-3-ol

To a solution of ((3R,4S)-4-azidotetrahydrofuran-3-yloxy)trimethylsilane(7.80 g, 38.8 mmol) in MeOH (100 mL), was added TFA (10.0 mg, 0.002equiv), and the mixture was allowed to stir at room temperature for 30min. The resulting solution was treated with Pd/C (1.90 g, 25 wt %) andallowed to stir at ambient temperature under H₂ atmosphere for 40 h. Thereaction solution was filtered through Celite and the filter cake waswashed with MeOH. The combined organics were concentrated under reducedpressure to afford the title compound (3.10 g). ¹H NMR (CDCl₃, 400 MHz):δ 3.38-3.40 (m, 2H), 3.54 (m, 2H), 3.68-3.72 (m, 2H), 3.75 (dd, 1H),3.83 (dd, 1H), 4.00-4.03 (m, 1H).

Step 3: Intermediate 4, tert-butyl (3S,4R)-4-hydroxytetrahydrofuran-3-ylcarbamate

To a solution of (3R,4S)-4-aminotetrahydrofuran-3-ol (1.00 g, 9.71 mmol)in THF (30 mL) was added (BocfiO (2.70 g, 1.30 equiv). The mixture wasallowed to stir at ambient temperature overnight. The reaction solutionwas concentrated under reduced pressure. The residue was partitionedbetween EtOAc and water, the organic layer was washed with water andbrine, dried over anhydrous Na₂SO₄ and concentrated under reducedpressure. The crude product was purified by column chromatography(eluting with 5% EtOAc/hexane) to afford the title compound as a whitesolid (1.20 g, 61%). ¹H NMR (CDCl₃, 400 MHz): δ 1.45 (s, 9H), 3.11 (s,1H), 3.60-3.63 (m, 1H), 3.68-3.71 (1H), 3.95 (s, br, 1H), 4.04-4.11 (m,2H), 4.28-4.30 (m, 1H), 4.74 (s, br, 1H).

Step 4: Intermediate 5,(3R,4S)-4-(tert-butoxycarbonylamino)tetrahydrofuran-3-ylmethanesulfonate

To the solution of tert-butyl (3S,4R)-4-hydroxytetrahydrofuran-3-ylcarbamate (1.20 g, 5.91 mmol) and TEA (0.89 g, 1.50 eq) in DCM (40 mL),was added methanesulfonyl chloride (0.88 g, 1.30 eq), in portions at 0°C. under a nitrogen atmosphere. The mixture was slowly warmed to roomtemperature and was stirred for 1 h. The resultant solution was washedwith water, 1 N HCl, and saturated aqueous sodium bicarbonate, driedover anhydrous Na₂SO₄ and concentrated under reduced pressure to affordthe title compound (1.60 g, 96%). ¹H NMR (CDCl₃, 400 MHz): δ 1.45 (s,9H), 3.21 (s, 3H), 3.70-3.73 (m, 1H), 3.97-3.99 (m, 1H), 4.05-4.07 (m,1H), 4.17-4.19 (m, 2H), 4.76 (s, 1H), 5.05 (d, 1H).

Step 5, Intermediate 6, tert-butyl (3R,4S)-4-azidotetrahydrofuran-3-ylcarbamate

To a solution of(3R,4S)-4-(tert-butoxycarbonylamino)tetrahydrofuran-3-ylmethanesulfonate (1.60 g, 5.70 mmol) in NMP (10 mL), was added NaN₃(0.92 g, 2.50 eq). The mixture was stirred at 95° C. for 5 h. Theresultant solution was quenched with water and extracted with ethylacetate. The organic phase was washed with brine, dried over sodiumsulfate, filtered, and concentrated under reduced pressure to afford thetitle compound (620 mg, 48%). ¹H NMR (CDCl₃, 400 MHz): δ 1.47 (s, 9H),3.45 (t, 1H), 3.87-3.89 (m, 1H), 4.02-4.07 (m, 2H), 4.21 (s, br, 1H),4.36-4.44 (m, 1H), 4.85 (s, br, 1H).

Step 6: Intermediate 7, tert-butyl (3R,4S)-4-aminotetrahydrofuran-3-ylcarbamate

To a solution of tert-butyl (3R,4S)-4-azidotetrahydrofuran-3-ylcarbamate(620 mg, 2.72 mmol) in methanol (15 ml), was added Pd/C (10%, 170 mg).The mixture was hydrogenated (3 atm) at ambient temperature underhydrogen overnight, after which the reaction mixture was filtered andthe filtrate concentrated to afford the title compound (350 mg, 63%, 91%ee). ¹H NMR (CDCl₃, 400 MHz): δ 1.26 (s, br, 1H), 1.46 (s, 9H), 1.68 (s,br, 1H), 3.47-3.49 (m, 1H), 3.57-3.59 (m, 2H), 3.98-4.06 (m, 2H), 4.10(d, 1H), 5.24 (s, br, 1H).

Intermediate 7 was used to make I-241, by coupling with Intermediate 8from Example 224 followed by Boc deprotection and acrylamide formationusing the procedure described in Example 224. I-241: MS m/z: 520.4(M+H⁺), ¹H NMR (400 MHz, CDCl₃): 3.68-3.85 (m, 5H), 3.95 (s, 6H),4.16-4.24 (m, 2H), 4.87 (br, 2H), 5.62 (d, 1H), 6.02-6.08 (m, 1H),6.24-6.39 (m, 3H), 6.63 (s, 1H), 7.43 (s, 1H), 8.47 (s, 1H).

Example 227: Synthesis of I-242

Step 1: Intermediate 1

To a solution of 4-methoxy-2-nitroaniline (170 mg, 1.02 mmol) inanhydrous NMP (5 mL) was added NaH (60%, 42.0 mg, 1.02 mmol). Thereaction mixture was stirred for 10 min at room temperature after whichit was heated at 100° C. for 0.5 h, followed by addition of CommonIntermediate 8 from Example 223 (300 mg, 0.68 mmol). The reactionmixture was allowed to stir at 100° C. overnight after which it wascooled to room temperature and partitioned between EtOAc and water. Theorganic phase was separated and washed with water, brine, dried overanhydrous Na₂SO₄ and the crude product was purified through silica gelcolumn chromatography to afford the title compound (200 mg, 55%). MSm/z: 532.4 (M+H⁺).

Step 2: Intermediate 2

A mixture of Intermediate 1 (200 mg, 0.38 mmol), Fe (130 mg, 2.26 mmol),and NH₄Cl (130 mg, 2.43 mmol), in EtOH (10 mL) with H₂O (6 mL), washeated to reflux for 1 h. The resultant solid was removed by filtrationand the filtrate was extracted with DCM. The organic phase was washedwith brine, dried over Na₂SO₄ and the crude product purified throughsilica gel column chromatography to afford the title compound (180 mg,95%). MS m/z: 502.4 (M+H⁺).

Step 3: I-242

To a solution of Intermediate 2 (180 mg, 0.36 mmol) and DIPEA (70 mg,0.54 mmol) in anhydrous DCM (5 ml) at 0° C., was added a solution ofacryloyl chloride (40 mg, 0.43 mmol) in anhydrous DCM (1 mL) dropwise.After 10 min., the reaction mixture was partitioned between DCM/H₂O andthe organic phase was separated, washed with brine, and dried overNa₂SO₄. The resultant solid was purified through column chromatographyon silica gel to afford the title compound (60.0 mg, 30%). MS m/z: 556.4(M+H⁺). ¹H NMR (400 Hz, DMSO-d₆): δ 3.48 (s, 3H), 3.78 (s, 3H), 3.96 (q,6H), 5.74 (d, 1H), 6.27 (dd, 1H), 6.53 (q, 1H), 6.82 (dd, 1H), 6.99 (s,1H), 7.40 (br, 1H), 7.58 (br, 1H), 7.76 (s, 1H), 8.73 (s, 1H), 9.15 (s,1H), 9.68 (br, 1H).

Example 228: Synthesis of I-243

The title compound was prepared as described in Example 227 using4-methyl-2-nitroaniline in place of 4-methoxy-2-nitroaniline in Step 1.MS m/z: 540.5 (M+H⁺).

Example 229: Synthesis of I-244

The title compound was prepared as described in Example 227 using2-nitroaniline in place of 4-methoxy-2-nitroaniline in Step 1. MS m/z:526.4 (M+H⁺).

Example 230: Synthesis of I-245

The title compound was prepared as described in Example 227 using4-fluoro-2-nitroaniline in place of 4-methoxy-2-nitroaniline in Step 1.MS m/z: 544.4 (M+H⁺).

Example 231: Synthesis of I-246

The title compound was prepared as described in Example 127 using4-chloro-3-(7-chloro-1-methyl-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-N-(3-(2-cyanopropan-2-yl)phenyl)benzamidein place of Intermediate 6 in Step 5 (which was prepared as described inExample 1 using3-amino-4-chloro-N-(3-(2-cyanopropan-2-yl)phenyl)benzamide in place of3,5-dimethoxyaniline in Step 4. MS m/z: 615.6 (M+H⁺).

Example 232: Synthesis of I-247

The title compound was prepared as described in Example 127 usingN-(3-(tert-butyl)phenyl)-4-chloro-3-(7-chloro-1-methyl-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)benzamidein place of Intermediate 6 in Step 5 (which was prepared as described inExample 1 using 3-amino-N-(3-(tert-butyl)phenyl)-4-chlorobenzamide inplace of 3,5-dimethoxyaniline in Step 4. MS m/z: 604.6 (M+H⁺).

Example 233: Protein Mass Modification Assays

An FGFR4 intact protein (from either SignalChem (method a in Table 6) orInvitrogen (method b in Table 6)), and a compound of the invention(10-fold excess of Compound to protein) were incubated for 60 min. Afterincubation, 5 μL aliquots of the samples were diluted with 15 μL of 0.2%TFA prior to desalting using a micro C₄ ZipTip protocol, which was addeddirectly onto the MALDI target using sinapinic acid as the desorptionmatrix (10 mg/mL in 0.1% TFA:Acetonitrile 50:50, v/v). The centroid massof FGFR4 in the control sample was compared with the centroid mass ofFGFR4 incubated with the compound of the invention. A shift in thecentroid mass of the treated FGFR4 compared to the untreated FGFR4 wasdivided by the molecular weight of the compound of the invention. Thiscalculation provided the percentage of modified protein after one hourincubation. This assay confirms whether or not the FGFR4 target iscovalently bound to a test compound (i.e., whether the protein's mass ismodified).

For example, to assess mass modification of FGFR4 with I-1, intact FGFR4(Invitrogen, Cat. No.: P3054) was incubated alone and with I-1 (10-foldexcess of I-1 to protein). After 60 minutes, the protein samples werediluted and prepared as described above. The centroid mass of theprotein (m/z: 42761.7; shown in FIG. 1, panel A) was compared with thecentroid mass of the treated protein (m/z: 43350.2; shown FIG. 1, panelB). The centroid mass shift of 589 Da (87%), indicated completemodification of FGFR4 by I-1. Other compounds also were tested in thisfashion. The results of these experiments are depicted in Table 6.

Tables 6, 7, and 8 show the activities of selected compounds of thisinvention in various FGFR assays. Compound numbers in Tables 6, 7 and 8correspond to the Compound numbers above.

Compounds having an activity designated as “A” provided anEC₅₀/IC₅₀/GI₅₀≤100 nM; compounds having an activity designated as “B”provided an EC₅₀/IC₅₀/GI₅₀ of 101-500 nM; compounds having an activitydesignated as “C” provided an EC₅₀/IC₅₀/GI₅₀ of 501-999 nM; compoundshaving an activity designated as “D” provided an EC₅₀/IC₅₀/GI₅₀ of ≥1000nM.

Compounds having an activity designated as “E” provided a massmodification of ≥70%; compounds having an activity designated as “F”provided a mass modification of 31-69%; compounds having an activitydesignated as “G” provided a mass modification ≤30%. Table 6: Summary ofMass Modification Data for Test Compounds

TABLE 6 Summary of Mass Modification Data for Test Compounds MS CMPDFGFR4 Method I-1 E a I-2 E a I-3 F a I-5 E a I-7 F a I-8 F a I-9 F aI-10 G a I-11 F a I-12 E a I-13 E a I-14 E a I-15 E a I-17 E a I-19 E aI-20 F a I-21 F a I-23 E a I-24 E a I-25 E a I-26 E a I-28 F a I-30 F aI-31 E a I-32 F a I-33 F a I-34 E a I-35 E a I-36 F a I-38 E a I-42 F aI-43 E b I-44 E b I-45 F b I-46 G a I-47 E a I-48 E a I-49 E a I-50 E aI-52 F a I-54 F a I-55 F a I-57 F a I-58 F a I-64 E b I-66 G b I-69 E bI-82 F b I-83 E b I-117 E b I-155 E a I-179 E a I-180 F a I-181 E aI-232 G a I-233 G a I-236 G a I-237 G a I-238 G a

Example 234: Omnia Assay Protocol for Potency Assessment Against FGFR 4Enzyme

A 10× stock solution of FGFR4-WT (PR4380C), (from Invitrogen, Carlsbad,Calif.) corresponding to method a in Table 7, was prepared as describedbelow. Alternatively, a 10× stock solution of FGFR4-WT (F01-11G),(SignalChem, Richmond, BC) corresponding to method b in Table 7, wasprepared as described below. A solution of 1.4× ATP (AS001A) and 5×Tyr-Sox conjugated peptide substrate (KNZ3101) were prepared in IXkinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCk, 1 mMEGTA, 5 mM β-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2mM DTT (DS001 A). 5 μL of FGFR4 was pipetted into a Corning (#3574)384-well, white, non-binding surface microtiter plate (Corning, N.Y.),containing a 0.5 μL volume of 100% DMSO. The serially diluted compoundswere prepared on a Tecan EVO100. A second addition of 10 μl of Tyr-SoxFGFR4 substrate was added to each well and the kinase reactions werestarted with the addition of 35 μL of 1.4× ATP. The reactions weremonitored every 71 seconds for 240 minutes at λ_(ex)360/λ_(em)485 in aSynergy plate reader from BioTek (Winooski, Vt.). At the conclusion ofeach assay, progress curves from each well were examined for linearreaction kinetics and fit statistics (R², 95% confidence interval,absolute sum of squares). Initial velocity (0 minutes to ˜60 minutes)from each reaction was determined from the slope of a plot of relativefluorescence units vs time (seconds) and then plotted against inhibitorconcentration to estimate IC₅₀ from log[Inhibitor] vs Response (VariableSlope model in GraphPad Prism from GraphPad Software (San Diego,Calif.)).

Methods:

a) [FGFR4-WT]=10 nM, [ATP]=300 uM, [Y10-Sox]=10 uM (ATP K_(Mapp)˜300uM),b) [FGFR4-WT]=2.5 nM, [ATP]=250 uM, [Y10-Sox]=10 uM (ATP K_(Mapp)˜250uM),tools.invitrogen.com/content/sfs/manuals/omnia_kinase_assay_man.pdf

The results of these experiments, reported as IC₅₀, show the ability ofthe compounds of the invention to inhibit FGFR enzyme activity and aredepicted in Table 7.

TABLE 7 Summary of Enzymatic Data for Test Compounds FGFR4 Cmpd IC₅₀(nM) Method I-1 A a I-2 A b I-3 A b I-5 A a I-6 C b I-7 C b I-8 A b I-9A b I-10 D b I-11 C b I-12 B b I-13 B b I-14 A b I-15 A b I-17 A a I-18D b I-19 B a I-20 A b I-21 A a I-22 C a I-23 A b I-24 A b I-25 B b I-26A b I-27 A a I-28 A a I-29 B a I-30 A a I-31 A a I-32 A a I-34 A a I-35A a I-36 A a I-37 B a I-38 C a I-39 D a I-40 A a I-41 A a I-42 A a I-43A a I-44 A a I-45 D a I-46 D a I-47 A a I-48 A a I-49 A a I-50 A a I-51D a I-52 C a I-53 A a I-54 A a I-55 A a I-56 A a I-57 A a I-58 A a I-59A a I-60 A a I-61 A a I-62 A a I-63 A a I-64 A a I-65 A a I-66 D a I-67A a I-68 B a I-69 A a I-70 A a I-73 B a I-74 A a I-75 A a I-76 C a I-77A a I-78 A a I-79 B a I-80 B a I-81 B a I-82 A a I-83 B a I-84 C a I-85A a I-86 A a I-87 A a I-88 A a I-89 A a I-90 A a I-91 A a I-92 A a I-93D a I-94 D a I-95 A a I-96 D a I-97 A a I-98 A a I-99 A a I-100 A aI-101 B a I-102 A a I-103 A a I-104 A a I-105 B a I-106 B a I-107 D aI-108 A a I-109 A a I-110 A a I-111 C a I-112 A a I-113 A a I-114 A aI-115 B a I-116 A a I-117 A a I-118 D a I-119 D a I-120 D a I-121 A aI-122 B a I-123 A a I-124 A a I-125 B a I-126 A a I-127 A a I-128 B aI-129 A a I-130 A a I-131 A a I-132 A a I-133 A a I-134 A a I-135 A aI-136 A a I-137 A a I-138 B a I-139 A a I-140 B a I-141 A a I-142 A aI-143 A a I-144 A a I-145 A a I-146 A a I-147 A a I-148 A a I-149 A aI-150 A a I-151 A a I-152 A a I-153 A a I-154 B a I-155 A a I-181 A bI-185 D a I-186 A a I-196 A a I-198 A a I-200 A a I-201 A a I-202 A aI-204 A a I-205 B a I-206 A a I-207 D a I-208 C a I-209 D a I-210 A aI-211 A a I-212 A a I-213 A a I-214 A a I-215 A a I-216 A a I-217 A aI-218 D a I-219 B a I-220 D a I-221 D a I-222 D a I-223 A a I-228 B aI-232 A b I-233 A b I-234 B b I-235 B b I-236 B b I-237 B a I-238 A aI-241 A a I-240 D a I-242 A a I-243 A a I-244 A a I-245 A a I-246 B aI-247 B a

Example 235: FGFR4 Signaling

Preparation of Cells: MDA-MB-453 (breast carcinoma) and Huh7(hepatocellular carcinoma) cells were used. Huh7 cells were grown inDMEM (Invitrogen, Carlsbad, Calif.) supplemented with 10% FBS(Invitrogen) and 1% Penicillin-Streptomycin (P/S, Lonza, Walkersville,Md.). MDA-MB-453 cells were grown in complete RPMI 1640 (Invitrogen),supplemented with 10% FBS and 1% P/S. All cells were maintained andpropagated as monolayer cultures at 37° C. in a humidified 5% CO₂incubator.

For the MSD and ELISA assays, total FGFR4 antibodies were obtained fromR&D Systems (Minneapolis, Minn.) and used at 1:500. For theimmunoblotting (Western blotting) assay, total FGFR4 antibody wasobtained from Santa Cruz (Santa Cruz, Calif.) and used at 1:1000.Phospho-FGFR antibodies were obtained from Cell Signaling (Danvers,Mass.) or R&D Systems and used at 1:1000. The phopho-FGFR antibody fromCell Signaling was used for immunoblotting, whereas the phopho-FGFRantibody from R&D was used for MSD and ELISA assays. Secondaryantibodies were used at 1:10,000. The goat anti-mouse IgG IRDye 800CWantibody was obtained from LiCor Biosciences (Lincoln, Nebr.) and thegoat anti-rabbit IgG Alexa Fluor 680 was obtained from Invitrogen. Theanti-rabbit Sulfo-tag and anti-streptavidin Sulfo-tag antibodies wasobtained from Meso Scale Discovery (Gaithersburg, Md.), and were used at1:1000 and 1:5000, respectively.

Immonoblotting (Western Blotting, WB)—Method A (Only for MDA-MB-453)

For MDA-MB-453 cells signaling, cells were grown in 96-wellpoly-D-lysine plates (BD Bioscience, San Jose, Calif.) to 90%confluence, and were then incubated in low-serum (0.1% FBS) media for16-18 hr. The cells were then treated with 5, 1.25, 0.31, 0.078, 0.020or 0.005 of test compound in low-serum (0.1% FBS) media for 1 hr. Aftertreatment, the cells were washed with cold PBS (Invitrogen) and wereimmediately lysed by freeze/thawing 3×in 32 μL of cold Cell ExtractionBuffer (Invitrogen) which was supplemented with Complete Proteaseinhibitors (Roche, Indianapolis, Ind.) and PhosphoSTOP (Roche)phosphatase inhibitors.

The MDA-MB-453 protein concentrations were determined by a BCA Assay(Pierce, Rockford, Ill.). A sample of 50-100 μg of each lysate wasseparated by a 4-12% gradient (SDS-PAGE (Invitrogen)), transferred to anitrocellulose membrane (Biorad, Hercules, Calif.), and probed withspecific antibodies. Phospho-protein signals were quantitated usingOdyssey Infrared Imaging (Li-Cor Biosciences).

To assess phospho-FGFR signaling, the blots were probed withanti-Phospho-FGFR (Y653/Y654) and total anti-FGFR antibodies. Thephospho-FGFR signal was normalized to total FGFR expression for eachsample. The results are indicated as % DMSO control. The normalized datawas fitted using a sigmoidal curve analysis program (Graph Pad Prismversion 5) with variable Hill slope to determine the EC₅₀ values. Theresults are provided in Table 8, under the column titled “Signaling EC₅₀(nM)”.

Meso Scale Assay (MSP)—Method B (for Both MDA-MB-453 and Huh7)

MDA-MB-453 and Huh7 cells were grown in 96-well poly-D-lysine plates (BDBioscience, San Jose, Calif.) to 90% confluence. The cells were thenincubated in low-serum (0.1% FBS) media for 16-18 hr. and then treatedwith 5, 1.67, 0.56, 0.185, 0.068, 0.021 or 0.007 μM of test compound inlow-serum (0.1% FBS) media for 1 hr. After treatment, the cells werewashed with cold PBS (Invitrogen), and were immediately lysed byfreeze/thawing 3× in 32 μL of cold Cell Extraction Buffer (Invitrogen),which was supplemented with Complete Protease inhibitors (Roche) andPhosphoSTOP (Roche) phosphatase inhibitors.

MSD plates (Meso Scale Discovery) were coated with total FGFR-4antibodies overnight at 4° C. A lysate (25 μL) was added to the MSDplate overnight at 4° C. The MSD signals were obtained by incubatingwith a phospho-FGFR antiobody (R&D Systems) and an anti-rabbit sulfo-tagantibody (Meso Scale), for 2 hr at room temperature. The results areindicated as % DMSO control. The data was fitted using a sigmoidal curveanalysis program (Graph Pad Prism version 5) with variable Hill slope todetermine the EC₅₀ values. The results are provided in Table 8, underthe column titled “Signaling EC₅₀ (nM)”.

ELISA—Method C (Only for MDA-MB-453)

MDA-MB-453 cells were grown in 96-well poly-D-lysine plates (BDBioscience, San Jose, Calif.) to 90% confluence. The cells were thenincubated in low-serum (0.1% FBS) media for 16-18 hr. The MDA-MB-453cells were then treated with 5, 1.67, 0.56, 0.185, 0.068, 0.021 or 0.007μM of test compound in low-serum (0.1% FBS) media for 1 hr. Aftertreatment, the MDA-MB-453 cells were washed with cold PBS (Invitrogen)and were immediately lysed by freeze/thawing 3× in 32 μL of cold CellExtraction Buffer (Invitrogen), which was supplemented with CompleteProtease inhibitors (Roche) and PhosphoSTOP (Roche) phosphataseinhibitors.

Nunc-immuno plates (96-well; Sigma, St. Louis, Mo.) were coated withtotal FGFR-4 antibodies overnight at 4° C. A lysate (25 μL) was added tothe plate for 2 hr at room temperature. A phospho-FGFR detectionantibody (100 μL) was added per well for 2 hr at room temperature,followed by a goat anti-rabbit HRP antibody (100 μL) for 45 min. Theresults are indicated as % DMSO control. The data was fitted using asigmoidal curve analysis program (Graph Pad Prism version 5) withvariable Hill slope to determine the EC₅₀ values.

The results of these experiments show the ability of the compounds ofthe invention to inhibit phosphor-signaling of FGFR4 in cells. Theresults are depicted in Table 8 under the column titled “Signaling EC₅₀(nM)”. MDA-MB-453 cell results fall under the heading “pFGFR4 (MDA)” andHuh7 results fall under the heading “pFGFR4 (Huh)”

Example 236: Cell Proliferation

MDA-MB-453 and Huh7 cells were plated in the appropriate Growth Mediasupplemented with 5% FBS and 1% P/S in a 96 well tissue culture plates(Corning), as indicated in Table 8. For both MDA-MB-453 and Huh7 cellsthe starting density was 5000 cells per well. The cells were allowed tosettle down for 4 hr and were then treated with 5, 1.25, 0.31, 0.078,0.020 or 0.005 μM of test compound for: 96 hr for MDA-MB-453 and 120 hrfor Huh7 cells. Cell viability was determined by CellTiter Glo (Promega,Madison, Wis.) and the results were converted to cell numbers using astandard curve. The growth inhibition (GI₅₀) values were determined byGraph Pad Prism.

The results of these experiments show the ability of compounds toinhibit cell growth in FGFR dependant cell lines and are depicted inTable 8, in the columns titled “Cell Proliferation GI₅₀ (nM)”.MDA-MB-453 cell results fall under the heading “MDA” and Huh7 cellresults fall under the heading “Huh.”

Example 237: Target Occupancy Assay

In order to assess free FGFR4 protein a biotinylated covalent probe wasutilized. As described in Example 235, Method B, MDA-MB-453 cells weretreated with test compound, washed, and lysed. Each lysate (25 μL) wasadded to a 96-well plate and 2 μM of a biotinylated covalent probe(I-127) was added. The reaction was incubated for 2 hr at roomtemperature. The samples and probe mixture were transferred to aFGFR4-coated MSD plate for 2 hr at room temperature. MSD signals wereobtained with an anti-streptavidin Sulfo-tag (Meso Scale) antibody for 1hr at room temperature. The results are indicated as % DMSO control. Thedata was fitted using a sigmoidal curve analysis program (Graph PadPrism version 5) with variable Hill slope to determine the EC₅₀ values.

The results of these experiments show the ability of the compounds ofthe invention to covalently modify FGFR4 in MDA-MB-453 cells byassessing the amount of free FGFR4 protein. If a compound completely(100%) covalently modes FGFR4 no free FGFR4 should be available forcovalent modification by the biotinylated probe and subsequent bindingto streptavidin and detection. Results are depicted in Table 8, in thecolumn titled “FGFR4 Occupancy EC₅₀ (nM)”.

TABLE 8 Summary of Cellular Data for Test Compounds Method for SignalingCell FGFR4 Signaling EC₅₀ (nM) Proliferation Occupan- EC₅₀ De- Com-pFGFR4 pFGFR4 GI₅₀ (nM) cy EC₅₀ termination pound (MDA) (Huh) MDA HUH(nM) MDA I-1 A A A B A B I-2 A A B A I-3 A A A A C I-5 B B C D B B I-6 BB A B B I-7 C B I-8 A B A B B B I-9 A B A C A B I-10 D B I-11 D B I-12 AB B B I-13 B B B D B B I-14 B B I-15 A A B B I-17 A B B B I-18 D B I-19B B B C C B I-20 A A B B I-21 A A B C B B I-22 D B I-23 A A B I-24 A A BI-25 A B A C B B I-26 A A A A A B I-28 A B I-30 B B I-31 B B B I-32 A CB I-33 B D D B I-34 A C B I-35 B B C B B I-36 B B D B I-38 D B I-40 D BI-41 B B D B I-42 B A D B I-43 C D B B I-44 A A B D B B I-45 D B I-46 DB I-47 B C D B I-48 B B C D B I-49 C B I-50 B B D B I-52 D B I-54 B BI-55 A B B B B I-56 A B I-57 A B I-58 B B I-59 B B C D B B I-60 B B I-61B B B C B B I-62 A B I-63 B A B B B B I-64 B B B D B I-65 B B I-66 D BI-67 A C B I-68 B C D D B I-69 A B B C B I-70 B B I-73 B B I-74 A B I-75A B B I-76 C D D B I-77 A B B I-78 B B B I-79 B C B I-80 B D D B I-81 CD D B I-82 A A B I-83 B B C D B I-84 B B I-85 A A B B I-86 A A B I-87 AA A B B I-88 B B C D B I-89 B B I-90 B B B I-91 A A B I-92 B B B I-93 DB I-94 D B I-95 B B B C B B I-96 D B I-97 B B B D B I-98 A A B D B I-99B B B D B I-100 A A B C B I-101 B B I-102 B A D B I-103 A A B B I-104 AA A B B I-105 B B C B I-106 B B D B I-107 C D D B I-108 A B B I-109 A AB I-110 A A B C B I-111 C D D B I-112 B B I-113 B B C B I-114 B B B B BB I-115 B B D B I-116 A A A B B I-117 A A B B I-118 D B I-119 D B I-120D B I-121 A A B C B I-122 B B I-123 A B I-124 A A A B B I-125 C D D BI-126 A A A B A B I-127 B B D B I-128 D B I-129 B B I-130 A B I-131 A AA B B I-132 B B D B I-133 A A A B B I-134 A A A B I-135 A A B I-136 A AB I-137 B B C B I-138 B B D B I-139 B B C B I-140 B B C B I-141 A BI-142 B B I-143 B B B I-144 A B I-145 A B I-146 B B D B I-147 A B I-148A B I-149 A A B I-150 A B I-151 C D D B I-152 A B I-153 A A B B I-154 BD D B I-155 B D B I-179 A B B A I-180 A C I-181 A B I-185 B D D B I-186A A A B I-198 A B I-200 B I-201 B C I-204 A I-205 B I-206 A A I-207 DI-208 D I-209 D I-210 A B I-211 A A I-212 A A I-213 A A I-214 B B I-215A B I-216 B B I-217 A A I-218 D I-219 C I-220 D I-221 D I-223 A B I-228B B I-232 A A I-233 A A I-234 B D D A I-235 C B I-236 B D B I-237 B BI-238 A A A B I-241 A A I-240 D D I-242 A A I-243 A I-244 A I-245 AI-246 B I-247 A

Example 238: Washout Experiment

MDA-MB-453 cells were plated in the appropriate Growth Mediasupplemented with 10% FBS and 1% P/S, to 90% confluence in either 12- or−96 well tissue culture plates. The cells were allowed to settle downfor 4 hrs and were then maintained in low-serum (0.1% FBS) mediaovernight.

The following morning, the media was removed and the cells were treatedwith 1000-2000 nM of test compound in low-serum media for 1 hr. Thecells were washed free of test compound (3×) with PBS (Invitrogen). Oneset of cells was immediately lysed as indicated above as the 0 hr timepoint. The remaining cells were incubated with the appropriate completegrowth media (10-20% FBS) for 1, 2, 4, 8, 16 (in certain circumstances)and 24 hr. DMSO (0.5%) controls were collected at all time points.

Representative data is shown in FIGS. 2 and 3. The data in FIG. 2 showthat a representative compound, I-69, provides prolonged inhibition ofautophosphorylation of FGFR4 assessed by detection of phosphorylatedFGFR4 (pFGFR4) after cells are washed. The graph in FIG. 2 shows thatinhibition of pFGFR4 is consistent with the resynthesis rate of FGFR4 inthe cells examined (T1/2˜4-8 h). The data in FIG. 3 compares theduration of action of a covalently modifying (irreversible) compound,I-1, and its corresponding noncovalently modifying (reversible) analog,I-234. I-1 which covalently modifies FGFR4 has prolonged duration ofaction consistent with the resynthesis rate of FGFR4 in the cellswhereas I-234 does not show prolonged duration of action post washout.

While a number of embodiments of this invention are described herein, itis apparent that the basic examples may be altered to provide otherembodiments that utilize the compounds and methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments that have been represented by way of example.

1. A compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: X¹ is —CR⁴; X²is —CR⁵; X³ is N; X⁴ is N; X⁵ is C; G is H, O, OR, or N(R)(R); Ring A isan optionally substituted group selected from phenyl, a 3-8 memberedsaturated or partially unsaturated carbocyclic ring, a 5-6 memberedmonocyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, a 4-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, or a 7-10 membered bicyclicsaturated, partially unsaturated or aryl ring; each R is independentlyhydrogen or an optionally substituted group selected from C₁₋₆aliphatic, phenyl, a 3-8 membered saturated or partially unsaturatedcarbocyclic ring, a 4-7 membered heterocylic ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or a 5-6membered monocyclic heteroaryl ring having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur; or two R groups on the samenitrogen are taken together with the nitrogen atom to which they areattached to form a 4-7 membered heterocylic ring having 0-2 additionalheteroatoms independently selected from nitrogen, oxygen, or sulfur, ora 4-7 membered heteroaryl ring having 0-4 additional heteroatomsindependently selected from nitrogen, oxygen, or sulfur; R¹ is a warheadgroup -L-Y; wherein R¹ is attached to an atom adjacent to the atom whereT is attached, wherein: R¹ is selected from

L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain wherein Lhas at least one double bond and one or two methylene units of L areoptionally and independently replaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—,—SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, —C(O)O—, cyclopropylene, —O—,—N(R)—, or —C(O)—; or L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one alkylidenyl double bond andat least one methylene unit of L is replaced by —C(O)—, —NRC(O)—,—C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—,and one additional methylene unit of L is optionally replaced bycyclopropylene, —O—, —N(R)—, or —C(O)—; or L is a bivalent C₂₋₈ straightor branched, hydrocarbon chain wherein one methylene unit of L isreplaced by cyclopropylene and one additional methylene unit of L isreplaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—,—SO₂—, —OC(O)—, or —C(O)O—; or L is a bivalent C₂₋₈ straight orbranched, hydrocarbon chain wherein L has at least one triple bond andone or two methylene units of L are optionally and independentlyreplaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—,—SO₂—, —OC(O)—, or —C(O)O—; and Y is hydrogen, C₁₋₆ aliphatic optionallysubstituted with oxo, halogen, NO₂, or CN, or a 3-10 membered monocyclicor bicyclic, saturated, partially unsaturated, or aryl ring having 0-3heteroatoms independently selected from nitrogen, oxygen, or sulfur, andwherein said ring is substituted with 1-4 R^(e) groups; or L is acovalent bond, —CH₂—, —NH—, —C(O)—, —CH₂NH—, —NHCH₂—, —NHC(O)—,—NHC(O)CH₂OC(O)—, —CH₂NHC(O)—, —NHSO₂—, —NHSO₂CH₂—, or —SO₂NH—, and Y isselected from: (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN;or (ii) C2-6 alkenyl substituted with oxo, halogen, NO₂, or CN; or (iii)C2-6 alkynyl optionally substituted with oxo, halogen, NO₂, or CN; or(iv) a saturated 3-4 membered heterocyclic ring having 1 heteroatomselected from oxygen or nitrogen wherein said ring is substituted with1-2 R^(e) groups; or (v) a saturated 5-6 membered heterocyclic ringhaving 1-2 heteroatom selected from oxygen or nitrogen wherein said ringis substituted with 1-4 R^(e) groups; or

(vii) a saturated 3-6 membered carbocyclic ring, wherein said ring issubstituted with 1-4 R^(e) groups; or (viii) a partially unsaturated 3-6membered monocyclic ring having 0-3 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, wherein said ring is substituted with1-4 R^(e) groups; or (ix) a partially unsaturated 3-6 memberedcarbocyclic ring, wherein said ring is substituted with 1-4 R^(e)groups; or

 or (xi) a partially unsaturated 4-6 membered heterocyclic ring having1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur,wherein said ring is substituted with 1-4 R^(e) groups; or

(xiii) a 6-membered aromatic ring having 0-2 nitrogens wherein said ringis substituted with 1-4 R^(e) groups; or

 or (xv) a 5-membered heteroaryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, wherein saidring is substituted with 1-3 R^(e) groups; or

 or (xvii) an 8-10 membered bicyclic, saturated, partially unsaturated,or aryl ring having 0-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, wherein said ring is substituted with 1-4R^(e) groups; or L is a covalent bond, —C(O)—, —N(R)C(O)—, or a bivalentC₁₋₈ saturated or unsaturated, straight or branched, hydrocarbon chain;and Y is selected from: (i) C₁₋₆ alkyl substituted with oxo, halogen,NO₂, or CN; or (ii) C2-6 alkenyl optionally substituted with oxo,halogen, NO₂, or CN; or (iii) C2-6 alkynyl optionally substituted withoxo, halogen, NO₂, or CN; or (iv) a saturated 3-4 membered heterocyclicring having 1 heteroatom selected from oxygen or nitrogen wherein saidring is substituted with 1-2 R^(e) groups; or (v) a saturated 5-6membered heterocyclic ring having 1-2 heteroatom selected from oxygen ornitrogen wherein said ring is substituted with 1-4 R^(e) groups; or

 or (vii) a saturated 3-6 membered carbocyclic ring, wherein said ringis substituted with 1-4 R^(e) groups; or (viii) a partially unsaturated3-6 membered monocyclic ring having 0-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, wherein said ring issubstituted with 1-4 R^(e) groups; or (ix) a partially unsaturated 3-6membered carbocyclic ring, wherein said ring is substituted with 1-4R^(e) groups; or

 or (xi) a partially unsaturated 4-6 membered heterocyclic ring having1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur,wherein said ring is substituted with 1-4 R^(e) groups: or

 or (xiii) a 6-membered aromatic ring having 0-2 nitrogens wherein saidring is substituted with 1-4 R^(e) groups or

 or (xv) a 5-membered heteroaryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, wherein saidring is substituted with 1-3 R^(e) groups; or

 or (xvii) an 8-10 membered bicyclic, saturated, partially unsaturated,or aryl ring having 0-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, wherein said ring is substituted with 1-4R^(e) groups: each R^(e) is independently selected from -Q-Z, oxo, NO₂,halogen, CN, C₁₋₆ aliphatic optionally substituted with oxo, halogen,NO₂, or CN, or a suitable leaving group selected from alkoxy,sulphonyloxy, optionally substituted alkylsulphonyloxy, optionallysubstituted alkenylsulfonyloxy, optionally substituted arylsulfonyloxy,acyl, or diazonium, wherein: Q is a bivalent C₁₋₆ saturated orunsaturated, straight or branched, hydrocarbon chain, wherein one or twomethylene units of Q are optionally and independently replaced by—N(R)—, —S—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —SO—, —SO₂—, —N(R)C(O)—,—C(O)N(R)—, —N(R)SO₂—, or —SO₂N(R)—; and each Z is independentlyhydrogen or C₁₋₆ aliphatic substituted with oxo, halogen, NO₂, or CN;each R² is independently —R, halogen, -haloalkyl, —OR, —SR, —CN, —NO₂,—SO₂R, —SOR, —C(O)R, —CO₂R, —C(O)N(R)₂, —NRC(O)R, —NRC(O)N(R)₂, —NRSO₂R,or —N(R)₂; R³ is hydrogen, C2-6 alkenyl, —W-Cy, or C₁₋₆ alkyl, whereinthe C₁₋₆ alkyl is optionally substituted with 1-3 groups independentlyselected from halogen, —CN, oxo, —OR′, or —C(O)O(C₁₋₆ alkyl); W isabsent or is a bivalent C1-3 alkylene chain optionally substituted withone or more R″ and wherein one methylene unit of W is optionallyreplaced with —O—, —S—, or —NR′—; each R′ is independently hydrogen orC₁₋₆ alkyl; each R″ is independently halogen or C₁₋₆ alkyl, wherein theC₁₋₆ alkyl is optionally substituted with 1-3 groups independentlyselected from halogen, —CN, oxo, or —OR′; Cy is phenyl, C3-7 cycloalkyl,or a 3-7 membered monocyclic or 5-10 membered bicyclic saturated,partially unsaturated, or heteroaryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, wherein Cy isoptionally substituted with 1-3 R^(x); each R^(x) is independently H,—CN, oxo, —NH₂, C₁₋₆ alkyl, halogen, —OR′, —N(R′)₂, —NHC(O)(C₁₋₆ alkyl),—C(O)N(R′)₂, —C(O)O(C₁₋₆ alkyl), —NHSO₂(C₁₋₆ alkyl), or —SO₂N(R′)₂; orR³ is absent if not allowed by valence; R⁴ is hydrogen or an optionallysubstituted group selected from C₁₋₆ aliphatic, phenyl, a 3-8 memberedsaturated or partially unsaturated carbocyclic ring which is optionallybridged, a 4-7 membered heterocylic ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, a 5-6 memberedmonocyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, or a 7-10 membered bicyclic saturated,partially unsaturated or aryl ring, which is optionally bridged; R⁵ isindependently —R, halogen, —OR, —SR, —CN, —NO₂, —SO₂R, —SOR, —C(O)R,—CO₂R, —C(O)N(R)₂, —NRC(O)R, —NRC(O)N(R)₂, —NRSO₂R, or —N(R)₂; Y is O orNR^(a); R^(a) is hydrogen or an optionally substituted C₁₋₆ aliphaticgroup; T is a covalent bond or a bivalent straight or branched,saturated or unsaturated C₁₋₆ hydrocarbon chain wherein one or moremethylene units are optionally replaced by —O—, —S—, —N(R)—, —C(O)—,—OC(O)—, —C(O)O—, —C(O)N(R)—, —N(R)C(O)—, —N(R)C(O)N(R)—, —S(O)—, —SO₂—,—SO₂N(R)—, —N(R)SO₂—, or —N(R)SO₂N(R)—; and q is 0-6. 2-68. (canceled)69. The compound of claim 1, wherein G is H.
 70. The compound of claim69, wherein Ring A is phenyl, cyclohexyl, cyclohexenyl, cyclopentyl,cyclobutyl, cyclopropyl, pyridine, pyrimidine, pyrazine, pyridazine,pyrrole, pyrazole, piperidine, piperidin-one, pyrrolidine,tetrahydropyran, tetrahydrofuran, tetrahydrothiophene dioxide, orcyclobutene dione.
 71. The compound of claim 70, wherein R¹ is


72. The compound of claim 71, wherein R³ is hydrogen.
 73. The compoundof claim 72, wherein R⁴ is ethyl, phenyl, cyclohexyl,


74. The compound of claim 73, wherein R⁴ is


75. The compound of claim 74, wherein R⁴ is


76. The compound of claim 74, wherein R⁵ is H.
 77. The compound of claim76, wherein Y is NR^(a).
 78. The compound of claim 77, wherein R^(a) ishydrogen.
 79. The compound of claim 78, wherein T is a covalent bond.80. The compound of claim 79, wherein q is
 0. 81. The compound of claim1, where the compound is:

or a pharmaceutically acceptable salt thereof.
 82. A compositioncomprising a compound according to claim 1, and a pharmaceuticallyacceptable adjuvant, carrier, or vehicle.
 83. A method for inhibitingactivity of FGFR4, or a mutant thereof, in a patient or in a biologicalsample comprising the step of administering to said patient orcontacting said biological sample with a compound according to claim 1.84. The method according to claim 83, wherein the activity of FGFR4, ora mutant thereof, is inhibited irreversibly.
 85. The method according toclaim 84, wherein the activity of FGFR4, or a mutant thereof, isinhibited irreversibly by covalently modifying Cys 552 of FGFR4.
 86. Amethod for treating a FGFR4-mediated disorder in a patient in needthereof, comprising the step of administering to said patient a compoundaccording to claim
 1. 87. The method according to claim 86, wherein thedisorder is hepatocellular carcinoma or rhabdomyosarcoma.